Methods and kits for multiplexed fluorescence microscopy

Abstract

A method for multiplexed fluorescence microscopy is provided, comprising contacting a fixed sample with a set of binder-T oligonucleotide conjugates to bind the binder to any binding partner present in the sample, the set comprising multiple binders with different specificities, the sequence of the T oligonucleotide being unique to the binder to which it is conjugated; contacting the sample and any binder bound as a result of step a with a FRET oligonucleotide; irradiating the sample with a wavelength that causes excitation of the emitter molecule of the FRET oligonucleotide; and observing the fluorescence kinetic profile of the sample at the emission wavelength of the FRET oligonucleotide emitter molecule over time in one or more pixels, the FRET oligonucleotide being capable of hybridizing to multiple T oligonucleotides in the set to form multiple pairs, the dissociation and reassociation between each different pair generating a fluorescence kinetic profile unique to that pair within the set. Related kits, sets of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides, and methods for designing such sets are also provided.

Description

[Technical field]

[0001] The present invention is in the field of multiplexed fluorescence microscopy. [Background technology]

[0002] Fluorescence microscopy Fluorescence microscopy is a major component of life science and pharmaceutical research. Many areas of life science and pharmaceutical research rely heavily on fluorescence microscopy to observe biological samples at a subcellular level, allowing high-resolution imaging of intracellular components. Orthogonal labeling strategies also allow imaging of different components within the same cell using multiple channels (e.g., based on the use of multiple fluorophores), and these multichannel microscopy image configurations are extremely useful in a variety of downstream analyses.

[0003] To be able to image different channels from the same sample, one usually needs to utilize known excitation / emission spectra from various fluorescent proteins or dyes. The number of channels that can be imaged in a standard fluorescence microscopy setup is limited by the amount that the emission / excitation distributions of the available fluorophores overlap. In fact, fluorescence is notoriously difficult to multiplex, i.e., to measure multiple analytes simultaneously. In theory, any number of different fluorophores can be used in a fluorescence microscopy experiment, but when different fluorophores excite or emit with overlapping wavelengths of light, there is no way to deconvolute the fluorescent signals, or separate them into their respective channels. Thus, standard fluorescence microscopy is in practice limited to the use of 4-5 different fluorophores, each with significantly different excitation / emission wavelengths, to be able to assess 4-5 image modalities in a single sample. Thus, in practice, this spectral overlap issue limits the number of different components that can be visualized simultaneously.

[0004] Recent advances in single-molecule localization microscopy Oligonucleotide-based fluorescent labeling strategies have been developed that improve the resolution of fluorescence microscopy because they can overcome the Abbe diffraction limit. Briefly, techniques such as DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography), an example of a single-molecule localization microscopy (SMLM) technique (see, for example, Schnitzbauer, J., Strauss, MT, Schlichthaerle, T., Schueder, F., and Jungmann, R. (2017), Super-resolution microscopy with DNA-PAINT. Nat. Protocols 12, 1198-1228), use fluorescently tagged DNA probes that reversibly hybridize to a complementary docking strand tethered to a binding molecule (e.g., an antibody). When the binding molecule is stationary (e.g., because it is bound to a binding partner in a fixed cell sample), the reversible hybridization between the labeled strand and the docking strand results in a transient emission event. The transient emission event can be detected. The localization data generated can be used to create images of the sample at high resolution. The transient nature of the emission events allows for the temporal separation of molecules that could not otherwise be resolved spatially (e.g., because the molecules are located so close to each other that the signals are indistinguishable when attempting to image using standard techniques).

[0005] The ability to record movies capturing multiple stochastic binding events (e.g. at different sites in a sample) means that these events can ultimately be reconstructed into a single super-resolution image: each emission profile can be fitted with a point spread function that allows precise localization of single molecules with sub-diffraction-limited resolution.

[0006] A major limitation of the DNA-PAINT technique is the high background associated with the technique. This arises as a result of the use of fluorescently labeled imager probes. Fluorescence from these unbound probes contributes a large amount of background fluorescence. This high background not only creates problems in detecting the signal peaks of individual bound probe molecules, but also creates other practical limitations. Attempts to solve this problem of high background often result in a trade-off: reducing the concentration of the imager probe can reduce the background, but reducing the concentration of the imager probe reduces the rate at which complexes form (and therefore the frequency of binding events), thus limiting the rate at which image capture can occur and thus preventing rapid image capture. Thus, the image capture process with DNA-PAINT is often slow. Furthermore, SMLM is typically limited to a maximum of three laser wavelengths for excitation, making it typically not possible to image more than three modalities in a single experiment.

[0007] The inventors have devised a method for multiplexed fluorescence microscopy that can use a single wavelength excitation-illumination protocol. The method exploits the stochastic emission of fluorescence provided by DNA-based fluorescence strategies, and the use of Förster Resonance Energy Transfer (FRET) oligonucleotides allows for enhanced spectrally independent multiplexed modality imaging, as compared to imaging using, for example, oligonucleotide probes that are fluorescently labeled but may constantly emit a signal. As explained above, the use of standard fluorescent oligonucleotides in techniques such as DNA-PAINT is accompanied by a high background, given the continuous fluorescence of the oligonucleotides used. This high background is avoided in the method of the invention by using FRET oligonucleotides. These FRET oligonucleotides used in the invention have little or no background fluorescence when free in solution, but can fluoresce when hybridized with an oligonucleotide to form a binding pair. Avoiding high background means that a higher concentration of fluorescent oligonucleotides can be used in the method of the invention, and therefore the observation step (including image or video capture) is more rapid.

[0008] The principles of FRET and its use in conjunction with oligonucleotides are well known in the art. Such oligonucleotides use the principle of FRET using an emitter molecule and a quencher. Upon irradiation with the excitation wavelength of the emitter, if the emitter molecule and the quencher are located sufficiently close to each other (typically within 1-10 nm), no fluorescence is observed at the emission wavelength of the emitter. This is due to efficient energy transfer from the emitter molecule to the quencher. However, if the emitter molecule and the quencher are sufficiently physically separated, the rate of FRET is reduced, and thus emission from the emitter molecule can be detected at the emission wavelength of the emitter.

[0009] In the present invention, this principle is manipulated to generate a fluorescence kinetic profile. The FRET oligonucleotide comprises an emitter molecule and a quencher. When the FRET oligonucleotide is in solution, upon irradiation with the excitation wavelength of the emitter, no fluorescence is observed at the emission wavelength of the emitter due to efficient energy transfer to the proximally located quencher (e.g., due to a free / flexible conformation that allows the emitter molecule and quencher to be usually located at or near the two ends of the oligonucleotide in close proximity). The FRET oligonucleotide has the ability to reversibly hybridize with a member of a set of transactivating oligonucleotides (T oligonucleotides) conjugated to a binding agent. Upon hybridization with the T oligonucleotide, the structure of the FRET oligonucleotide is constrained by this hybridization. This physically separates the emitter molecule and the quencher, slowing down the rate of FRET and allowing emission from the emitter molecule at the emission wavelength of the emitter to be detected at the hybridization site upon irradiation with the excitation wavelength of the emitter. The T oligonucleotide and the FRET oligonucleotide are designed to allow reversible hybridization between the pairs formed. This means that for each pair a fluorescence kinetic profile is observed over time, i.e., fluorescence emission occurs when the binding pairs are in hybridized configuration, but not when the pairs are separated. As with DNA PAINT described above, the fact that a fluorescence kinetic profile is detected means that high resolution is achieved. Unlike DNA PAINT, background fluorescence is low, so the sensitivity or quality of the signal can be better than DNA PAINT, for example due to a higher signal-to-noise ratio.

[0010] Furthermore, the method and kit of the present invention allows multiplexing. Each FRET oligonucleotide can hybridize to multiple T oligonucleotides in a corresponding set, thus forming multiple pairs. Dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within that set. This means that multiple fluorescence kinetic profiles generated in a single set can be observed simultaneously in a single channel (i.e. based on the same emission spectrum), and the identity of the T oligonucleotide in each pair can be determined by its fluorescence kinetic profile (e.g. by calculating one or more metrics of the fluorescence kinetic profile that are uniquely attributed to that pair) since that fluorescence kinetic profile is unique to that pair within that set. Observation (e.g. detection or capture) of any fluorescence kinetic profile in a sample can be by recording a movie that captures the fluorescence kinetic profile at one or more, e.g. multiple, positions or pixels in the sample. This information is used to calculate one or more metrics of the fluorescence kinetic profile, which is then used to assign an identity to one or more pixels in the observed sample based on the metrics calculated from the fluorescence kinetic profile of the sample at that pixel, and finally to reconstruct one or more images of the sample or a portion thereof. A metric of a fluorescence kinetic profile is simply a measure or expression of one or more aspects of the fluorescence kinetic profile, and can be calculated or derived from the fluorescence kinetic profile, for example, by mathematical analysis of one or more aspects of the fluorescence kinetic profile.

[0011] The T oligonucleotide is conjugated to the binding agent such that the sequence of the T oligonucleotide is unique to the binding agent to which it is conjugated. Therefore, based on the fluorescence kinetic profile or its metrics observed at any given location in the sample, it is possible to determine which binding agent is present at that location. Non-limiting examples of these metrics include: (i) the average time between each fluorescence emission (also called off-time), (ii) the average duration of the fluorescence emission (also called on-time), and (iii) the incidence of the fluorescence emission. At least one, at least two, or at least three such metrics can be extracted from the fluorescence kinetic profile and used to describe it. Metrics other than these exemplary metrics may also be used.

[0012] The high resolution and low background achieved with this technique, combined with the inherent multiplexing capabilities, mean that this technique is particularly advantageous.

[0013] Using a single FRET oligonucleotide and a set of T oligonucleotides, multiple different fluorescence kinetic profiles (e.g., up to 10 or up to 25) can be generated. However, multiplexing is possible at multiple levels. At the first level, the present invention uses a FRET oligonucleotide that can bind to multiple T oligonucleotides in a set, so that each pair formed in the set generates a different fluorescence kinetic profile. Thus, by observing any fluorescence kinetic profile (or its metric) emitted from a sample over time, the identity of the T oligonucleotide that gives rise to any observed fluorescence kinetic profile, and therefore the specific binding partner to which the T oligonucleotide is conjugated, can be determined at any location in the sample.

[0014] Further multiplexing can be achieved by using multiple FRET oligonucleotides, each FRET oligonucleotide having a corresponding set of T oligonucleotides.By using two or more FRET oligonucleotides, each FRET oligonucleotide having a corresponding set of T oligonucleotides, a wider range of different fluorescence kinetic profiles can be generated, and therefore more binders can be used and more different target molecules can be detected.Therefore, this method is particularly advantageous for multiplexing.

[0015] As an example, the first set of T oligonucleotides and the corresponding FRET oligonucleotides may give rise to a number of different fluorescence kinetic profiles. Any number of numerical metrics or features may be extracted from the fluorescence kinetic profile. Non-limiting examples of these metrics include: (i) the average time between each fluorescence emission (also called off-time), (ii) the average duration of the fluorescence emission (also called on-time), and (iii) the incidence of the fluorescence emission. At least one, at least two, or at least three metrics may be extracted from the fluorescence kinetic profile and used to describe it, which may be the metrics listed above or alternative metrics.

[0016] Furthermore, the turning off and on of the fluorescent emission, conceptualized as "blinking," can be used to define a numerical metric that describes the hybridization affinity between each T oligonucleotide and the corresponding FRET oligonucleotide. From the blinking kinetics, at least (i) K on (association rate constant, expressed as M-1.s-1) and (ii) K offAny number of affinity metrics can be derived, including the dissociation rate constant (expressed as s-1). Since each T oligonucleotide is engineered to exhibit a unique hybridization kinetic profile, and therefore a unique blinking kinetic profile, any individual metric or combination of metrics can be used to distinguish the identity of the T oligonucleotide under observation. These affinity metrics can also be a way to describe the fluorescence kinetic profile.

[0017] A given set of T oligonucleotides may, for example, include 10 T oligonucleotides (T oligonucleotides 1-10). FRET oligonucleotide A may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 1-10). For example, by adding an additional set of T oligonucleotides including 10 T oligonucleotides (T oligonucleotides 11-20) and the corresponding FRET oligonucleotide (FRET oligonucleotide B), 10 more different fluorescence kinetic profiles (profiles 11-20) may be used.

[0018] A further degree of multiplexing is provided by using FRET oligonucleotides with different fluorophores that produce detectable fluorescent emissions in different spectral channels (e.g., using different fluorophores). Collecting signals in multiple spectral channels allows for a further level of multiplexing, allowing even more targets of binding agents to be detected in a single sample.

[0019] For example, a given set of T oligonucleotides may include, for example, 10 T oligonucleotides (T oligonucleotides 1-10). FRET oligonucleotide A may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 1-10). For example, by adding an additional set of T oligonucleotides including 10 T oligonucleotides (T oligonucleotides 21-30) and a corresponding FRET oligonucleotide (FRET oligonucleotide C) [FRET oligonucleotide C produces a fluorescence signal that is detectable in a different spectral channel than FRET oligonucleotide A (e.g., because FRET oligonucleotide C contains a different fluorophore than FRET oligonucleotide A)], 10 more fluorescence kinetic profiles (profiles 21-30) may be used. Because profiles 21-30 are detectable in a different spectral channel than profiles 1-10, one or more of the profiles may be similar or identical to one or more of profiles 1-10 because they are distinguishable based on being detected in different spectral channels (e.g., because FRET oligonucleotide C contains a different fluorophore than FRET oligonucleotide A). The T oligonucleotide remains unique to the binding partner, and each T oligonucleotide should give rise to a unique fluorescence kinetic profile within its detection channel.

[0020] Thus, unlike many other approaches, the ability to multiplex in this approach is not limited by the number of different fluorophores that can be spectrally resolved. Multiplexing in this method relies on the ability to generate distinguishable fluorescence kinetic profiles, which can be achieved by using differences in the base sequence of the T oligonucleotide. A large number of different T oligonucleotide and FRET oligonucleotide sequences can be designed and generated, meaning that there is an inherent multiplexing ability in the present invention even if only a single fluorophore is used (or even if the FRET oligonucleotide contains an emitter molecule with an overlapping emission spectrum), but further levels of multiplexing can be achieved by fluorophores that produce emissions that can be detected in different channels (e.g., using multiple fluorophores). The method of the present invention can also be easily integrated with standard microscopy setups, such as epifluorescence microscopes, confocal microscopes, total internal reflection fluorescence microscopes (TIRF), stimulated emission depletion (STED) microscopes, structured illumination microscopes (SIM), and other types of fluorescence microscopes that allow targeted excitation of fluorophore molecules contained within the sample by laser, LED, halogen, or other illumination sources.

[0021] Other techniques have attempted to multiplex DNA PAINT type probes, but generally they have achieved multiplexing using sequential binding of probes as described in WO2015 / 138653. The adaptability of the present technique to multiplexing is one of the advantages of the present method, along with low background fluorescence and improved imaging time. Due to these advantages, the method of the present invention is particularly applicable to high throughput assays. This utility in high throughput assays is particularly related to the ability to perform a high degree of multiplexing, which is desirable because more information is obtained in the experiment per experimental setup.

[0022] Thus, the method provided herein utilizes the fact that the base sequence of an oligonucleotide is a method of uniquely identifying the oligonucleotide. The base sequence also determines the hybridization properties of the oligonucleotide with other oligonucleotides. The base sequence of the T oligonucleotide and the corresponding FRET oligonucleotide can be programmed to generate pairs with specific fluorescence kinetic profiles, so that the identification of the fluorescence kinetic profile allows the identification of the specific T oligonucleotide present in the pair, and thus the identification of the specific binding partner to which the T oligonucleotide is conjugated. Summary of the Invention [Means for solving the problem]

[0023] The present invention is based on the inventors' discovery that the principle of FRET can be utilized in the context of a method of multiplexed fluorescence microscopy, using oligonucleotides to uniquely identify the binding partners to which they are conjugated, and these oligonucleotides are paired with FRET oligonucleotides. The use of FRET oligonucleotides has several advantages, as will be explained in more detail. First, when the FRET oligonucleotides are in solution (FIG. 1A), no fluorescence is observed at the emission wavelength of the emitter when illuminated with the excitation wavelength of the emitter molecule. This is due to efficient energy transfer from the emitter to the quencher, which is made possible by the free conformation of the FRET oligonucleotides in solution. This means that the background level of fluorescence (e.g., at the emission wavelength of the emitter) is low. In contrast, when the FRET oligonucleotides hybridize with complementary oligonucleotides immobilized throughout a cell sample, they emit fluorescence at the emission wavelength of the emitter (FIG. 1B). Hybridization of the FRET oligonucleotide with the T oligonucleotide linearizes the FRET oligonucleotide, which physically separates the quenching moiety from the emitting moiety, slowing down the rate of FRET. Thus, a fluorescent signal at the emission wavelength of the emitter is only emitted at the hybridization site and when the FRET oligonucleotide is hybridized. The sequences of the T oligonucleotide and the FRET oligonucleotide are designed so that the FRET oligonucleotide can hybridize to various T oligonucleotides in the set. Each different pair formed results in a unique fluorescence kinetic profile. This means that by observing the generated fluorescence kinetic profile and calculating distinguishable metrics of the fluorescence kinetic profile, it is possible to deconvolute the identity of the pair at a specific location in the sample (Figures 4 and 5), and thus determine the binding partner bound at that specific location in the sample.

[0024] Accordingly, the present invention provides a method for multiplexed fluorescence microscopy comprising the steps of: a. contacting the fixed sample with a set of binding agent-T oligonucleotide conjugates to allow the binding agent to bind any binding partner present in the sample, the set including multiple binding agents with different specificities, the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated; b. contacting the sample and any binding agent bound as a result of step a with a FRET oligonucleotide; c. illuminating the sample with a wavelength that causes excitation of the emitter molecule of the FRET oligonucleotide; d. observing a fluorescence kinetic profile at the emission wavelength of the FRET oligonucleotide emitter molecule over time in one or more pixels; wherein the FRET oligonucleotide can hybridize to a plurality of T oligonucleotides in the set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within the set.

[0025] The present invention also provides a kit for multiplexed fluorescence microscopy, comprising: a. a set of binding agent-T oligonucleotide conjugates, the set comprising multiple binding agents with different specificities, the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated; b. FRET oligonucleotides and wherein the FRET oligonucleotide is capable of hybridizing to a plurality of T oligonucleotides in the set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within the set.

[0026] The present invention also provides a set of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides for producing a kit for multiplexed fluorescence microscopy, comprising: a. a set of T oligonucleotides; b. FRET oligonucleotides and wherein the FRET oligonucleotide can hybridize to a plurality of T oligonucleotides in the set to form a plurality of pairs, and dissociation and reassociation between each different FRET oligonucleotide and T oligonucleotide produces a fluorescence kinetic profile that is unique for that pair within the set.

[0027] The present invention also provides a method of preparing the kit of the present invention, the method comprising providing a set of T oligonucleotides, and a set of binding agents and corresponding FRET oligonucleotides, and conjugating the set of T oligonucleotides with the set of binding agents to form a set of binding agent-T oligonucleotide conjugates, the set of binding agent-T oligonucleotide conjugates comprising a plurality of binding agents with different specificities, the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated within the set.

[0028] The invention further provides a method for designing a set of T oligonucleotides and FRET oligonucleotides for use in the methods of the invention, the method comprising: a. selecting a FRET oligonucleotide sequence at least 12 nucleotides in length; b. obtaining one or more sequences at least 8 nucleotides in length that are complementary to the FRET oligonucleotide sequence; c. generating a plurality of potential T oligonucleotide sequences, each of which differs by at least one nucleotide from the sequence complementary to the FRET oligonucleotide; d. selecting at least two T oligonucleotide sequences based on their ability to generate fluorescence kinetic profiles that differ from one another.

[0029] Multiplexed fluorescence microscopy Fluorescence microscopy Fluorescence microscopy is based on the detection of fluorescent compounds, which can be used to generate an image of a sample (this can be instead of or in addition to the detection of light). Fluorophores are fluorescent chemical compounds or biological proteins that can re-emit light upon photoexcitation. In fluorescence microscopy, light of one or more specific wavelengths is irradiated onto the fluorophores. This light is absorbed by the fluorophore(s) and the energy of the fluorophore is briefly raised to a higher excited state. The subsequent return to the ground state results in the emission of fluorescence that can be detected and measured. Detection of the emitted light can be used to generate an image.

[0030] Thus, a fluorophore is a molecule that emits light in response to light excitation, with the re-emitted light being at a longer wavelength than the light used for excitation. Any fluorophore has defined maximum excitation and emission wavelengths that correspond to the peaks of the excitation and emission spectra. Examples include fluorescein (FITC), rhodamine derivatives (TRITC), coumarin derivatives, and cyanine derivatives.

[0031] Fluorescence microscopy can be used, for example, to detect the presence or location of one or more target molecules in a sample. Certain target molecules, e.g., nucleic acids, can be detected using fluorescent stains, which can be small molecules that are intrinsically fluorescent. Nucleic acid stains such as DAPI and Hoechst are suitable examples that are routinely used to fluorescently label cell nuclei because they bind to the minor groove of DNA. To detect the presence or location of other target molecules in a sample, the ability of a specific binding agent to bind to the target molecule can be manipulated. For example, the use of antibodies as binding agents is well known. In such methods, an antibody against a specific target molecule can be used. A primary antibody (one that binds to the target molecule) can be labeled with a fluorescent molecule, or this fluorescent molecule can be carried by a second antibody that binds to the primary antibody. In this way, detection of fluorescence at a particular location in a sample indicates that the target molecule is present at that location.

[0032] As described in more detail below, the method involves observation of a fluorescent kinetic signal generated as a result of dissociation and reassociation between a FRET oligonucleotide and a T oligonucleotide, and therefore, fluorescence microscopy is used in the method.

[0033] multiplexing As used herein, multiplexing means that multiple binding agents are used, which generally means that multiple target molecules can be detected in the same sample if they are present (although multiple binding agents could, in principle, bind to a single target molecule, such as when multiple antibodies bind to a large polypeptide, each antibody binding to a different epitope). Thus, multiplexing can allow multiple target molecules to be observed in parallel.

[0034] These binding agents are generally contacted with the sample, for example, at the same time, and can bind to their respective targets if they are present in the same sample. One example of a standard multiplex assay is to use a first primary antibody against one target molecule and a second primary antibody against a second target molecule. The sample and any bound primary antibodies are then contacted with a corresponding secondary antibody that is labeled with multiple fluorophores, i.e., a first fluorophore against the first secondary antibody and a second fluorophore against the second secondary antibody. By detecting all the signals from each of the two fluorophores, an image of the location of the two target molecules in the sample can be generated.

[0035] Thus, the multiplexing process requires the ability to detect multiple fluorescent signals from the same sample. In standard fluorescence microscopy techniques, this can be done by using multiple fluorophores, for example fluorophores that emit light at sufficiently different wavelengths to be differentiated. A suitable example is the use of a fluorescein-based molecule as the first fluorophore and a rhodamine-based molecule as the second fluorophore. However, as mentioned above, multiplexing is difficult with fluorophores. The emission spectrum of most fluorophores is broad, limiting multiplexing to four or five labels in standard fluorescence microscopy. Furthermore, techniques that rely on the use of secondary antibodies also need to ensure that no cross-hybridization occurs between the secondary antibodies by using antibodies from different species. This limits the number of possible combinations, a limitation that is overcome in the present invention.

[0036] In the present invention, multiplexing is achieved by observing at least the presence or absence of a fluorescent signal at the emission wavelength of the relevant emitter molecule, as well as its temporal pattern. Rather than simply observing the presence or absence of fluorescence at the emission wavelength of the relevant emitter molecule, multiple "fluorescence kinetic profiles" are observed. The use of fluorescence kinetic profiles means that more information can be obtained from each channel used. Multiple binding agents with different specificities are used, each binding agent being conjugated to a T oligonucleotide. The sequence of the T oligonucleotide is unique to the binding agent to which it is conjugated. For a set of T oligonucleotides, there is a corresponding FRET oligonucleotide that can hybridize to multiple T oligonucleotides in the set to form multiple pairs. The sequences of the T oligonucleotides and the FRET oligonucleotides are such that the pairs formed undergo dissociation and reassociation, and this dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within the set. Because the fluorescence kinetic profile is unique to the pair within the set, observation of a particular fluorescence kinetic profile in one or more pixels indicates the presence of a particular T oligonucleotide at that location in the sample (and thus the presence of a binding agent to which the T oligonucleotide is conjugated, and ultimately the target molecule to which the binding agent binds, at that location in the sample). In other words, observation of a particular fluorescence kinetic profile in one or more pixels indicates the presence of a corresponding target molecule at that location in the sample.

[0037] The term "fluorescence kinetic profile" as used herein refers to the pattern of fluorescence emission resulting from dissociation and reassociation over time of any pair of a T oligonucleotide and its corresponding FRET oligonucleotide. Fluorescence from the emitter molecule (which is a direct result of the dissociation and reassociation of the pair over time) at the emission wavelength of the emitter molecule can be represented as fluorescence intensity over time (e.g., plotted as shown in Figures 3-5, 12, and 13). However, because the dissociation and reassociation of each pair is a stochastic process, it will be understood that individual observations of the fluorescence kinetic profile of a pair will not necessarily look the same every time, and each individual observation of the fluorescence kinetic profile of a given pair may look different when represented in a plot. However, there are recognizable characteristics or features of the fluorescence kinetic profile of a pair that are consistent between each individual observation of their fluorescence kinetic profile. These consistent characteristics or features can be used to describe and / or define the fluorescence kinetic profile of the pair. These characteristics or features include the metrics described elsewhere herein.

[0038] Thus, a single observed fluorescence kinetic profile may be described with reference to one or more consistent properties or characteristics (e.g., one or more of the metrics mentioned elsewhere herein). Thus, a single observed fluorescence kinetic profile of a pair may not necessarily look the same each time it is observed, but may be described as being specific to the pair within each set (or uniquely recognizable for the pair within each set), or unique within a set (or uniquely recognizable for the pair within a set), because the fluorescence kinetic profile of a given pair is unique and its consistent properties or characteristics allow for identification of the pairs within each set.

[0039] The method of the present invention is easily adapted to, and in fact is designed for, effective multiplexing, since a single FRET oligonucleotide can form multiple pairs, each of which generates a unique fluorescence kinetic profile, and each fluorescence kinetic profile is specific to, and therefore indicative of, the paired T oligonucleotide. In the observation stage, a fluorescence kinetic profile can be observed and recorded at one or more pixels. Where a fluorescence kinetic profile is observed, an identity can be assigned to each pixel based on the fluorescence kinetic profile. This is generally achieved by calculating a distinguishable metric of the fluorescence kinetic profile, and optionally assigning an identity to one or more pixels in the observed sample based on the metric of the fluorescence kinetic profile of the sample at that pixel. Multiplexing can also be achieved with a single FRET oligonucleotide, since each different T oligonucleotide-FRET oligonucleotide pair generates a unique fluorescence kinetic profile. In fact, using a single FRET oligonucleotide is advantageous, since multiple fluorescence kinetic profiles can be observed and, in some cases, recorded in the same spectral channel.

[0040] In the method of the present invention, the set of binding agent-T oligonucleotide conjugate generally comprises a plurality of binding agents with different specificity.For example, there can be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more binding agents with different specificity in the set, each binding agent is conjugated to T oligonucleotide, and the sequence of T oligonucleotide is specific to the binding agent that it is conjugated to. For example, there may be up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25 binding agents with different specificities in the set, each binding agent being conjugated to a T oligonucleotide, and the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated. Suitable ranges include 2-25, 3-24, 4-23, 5-22, 6-21, 7-20, 8-19, 9-20, 10-19, 11-18, 12-17, 13-16, 14-15 binding agents with different specificities in the set, each binding agent conjugated to a T oligonucleotide, and the sequence of the T oligonucleotide is unique to the binding agent to which it is conjugated.

[0041] By increasing the number of different FRET oligonucleotides used, more different fluorescence kinetic profiles can be achieved. This is because, even if a set of T oligonucleotides with highly diverse sequences is used, the number of different fluorescence kinetic profiles that can be generated for a given FRET oligonucleotide (i.e., a FRET oligonucleotide with a given sequence) may be finite. By increasing the number of different FRET oligonucleotides used in the method, each FRET oligonucleotide having a corresponding set of T oligonucleotides, the number of different fluorescence kinetic profiles that can be generated can be increased. Thus, more different binding agents can be used in the method of the present invention, and more target molecules can be detected in a single experiment.

[0042] As explained above, the average time between each fluorescence emission (which may be conceptualized as a "blink") is one component of the fluorescence kinetic profile and is a metric that may be derived from the observed fluorescence kinetic profile. When the T oligonucleotide and the FRET oligonucleotide hybridize or associate relatively quickly, i.e., when the duration that the two components are in a dissociated state from each other is short (short off time), a short average time between each fluorescence emission at the emission wavelength of the emitter molecule is observed. For example, in FIG. 3, profiles B and C represent pairs with shorter off times than profile A. When the T oligonucleotide and the FRET oligonucleotide hybridize or associate relatively quickly, but also dissociate relatively quickly, a high frequency or rate of "blinking" is observed. It may be possible to achieve a certain range of blinking kinetic profiles using a single FRET oligonucleotide and a set of T oligonucleotides. Using a second FRET oligonucleotide and a corresponding set of T oligonucleotides may allow the generation of fluorescence kinetic profiles that are different from those resulting from the first FRET oligonucleotide and the corresponding set of T oligonucleotides (e.g., they may produce a range that overlaps or does not overlap with the first range).

[0043] One aspect of the FRET oligonucleotide that can be easily varied (e.g., to increase the number of different fluorescence kinetic profiles) is the length of the FRET oligonucleotide. Longer oligonucleotides may hybridize more strongly to their pairs than shorter oligonucleotides, thereby increasing the average time it takes for the FRET oligonucleotide and the T oligonucleotide to dissociate. This increases the K off (e.g., a reduced K for a more strongly hybridizing pair than for a more weakly hybridizing pair. off (Values ​​can be seen). Thus, longer FRET oligonucleotides can contribute to pairs that produce fluorescence kinetic profiles with longer average durations of fluorescence emission (i.e., longer on-times) than pairs with shorter FRET oligonucleotides. For example, in FIG. 3, profile B is a representation of the fluorescence kinetic profile of a pair with a longer on-time than profiles A and C.

[0044] Having one longer FRET oligonucleotide and one shorter FRET oligonucleotide is just one example of an approach to generate an increased range of blinking kinetic profiles. Other examples include varying the base content (e.g., GC-rich FRET oligonucleotides tend to have a stronger chance of hybridizing to their pair than AT-rich oligonucleotides, thereby increasing the average time it takes for the FRET oligonucleotide and the T oligonucleotide to dissociate). This increases the K of the pair. off (e.g., a reduced K for a more strongly hybridizing pair than for a more weakly hybridizing pair. off values ​​can be seen).

[0045] Regardless of the exact method of generating multiple fluorescence kinetic profiles, it will be appreciated that by creating multiple sets of T oligonucleotides and corresponding FRET oligonucleotides, the number of different fluorescence kinetic profiles that can be generated will increase. Figure 3 illustrates this in a simplified form. Profiles B and C represent pairs with shorter off times than profile A, and profiles A and C represent pairs with shorter on times than profile B. It will be appreciated that many different fluorescence kinetic profiles can be generated, for example with different on and off time values. This increases the multiplexing possibilities. In other words, a higher degree of multiplexing is possible by using multiple sets of T oligonucleotides and corresponding FRET oligonucleotides than by using a single set of T oligonucleotides and corresponding FRET oligonucleotides. As will be described in more detail below, exemplary non-limiting metrics that can be derived or calculated from the fluorescence kinetic profile can include (i) the average time between each fluorescence emission (also called off time), (ii) the average duration of the fluorescence emission (also called on time), and (iii) the incidence rate of the fluorescence emission.

[0046] Illustrating the above, a given set of binder-T oligonucleotide conjugates may, for example, include 10 T oligonucleotides (T oligonucleotides 1-10). FRET oligonucleotide A may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 1-10). For example, by adding an additional set of binder-T oligonucleotide conjugates including 10 T oligonucleotides (T oligonucleotides 11-20) and corresponding FRET oligonucleotides (FRET oligonucleotides B), 10 more different fluorescence kinetic profiles (profiles 11-20) may be used. If FRET oligonucleotides A and FRET oligonucleotides B emit fluorescence that can be observed in the same channel (e.g., if they contain emitter molecules with overlapping emission spectra or if they contain the same emitter molecule), all 20 different fluorescence kinetic profiles may be observed in a single spectral channel. If FRET oligonucleotide A and FRET oligonucleotide B emit fluorescence that requires multiple spectral channels to observe the fluorescence kinetic profile, or if they can be or are optimally observed in different channels (e.g. if they contain emitter molecules with non-overlapping emission spectra, or if they contain emitter molecules with emission spectra with different emission maxima, or different emitter molecules), multiple spectral channels can be used to observe the fluorescence kinetic profile.

[0047] When multiple FRET oligonucleotides and corresponding T oligonucleotides are used, this is referred to as using a set of T oligonucleotides. Even when a set of T oligonucleotides is used, the sequence of each T oligonucleotide present is unique to the binding agent to which it is conjugated. Furthermore, the dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is (i) unique to that pair within that set, and (ii) unique to that pair within that set. Furthermore, in some embodiments, each T oligonucleotide hybridizes only to its corresponding FRET oligonucleotide, and does not hybridize to the FRET oligonucleotide(s) of other sets within that set. Cross-hybridization should be avoided so that the correlation between the fluorescence kinetic profile, the T oligonucleotide, and the binding agent is maintained.

[0048] Further levels of multiplexing can be achieved by increasing the number of different channels in which fluorescence kinetic signals are observed, for example by using FRET oligonucleotides that contain fluorophores with different emission spectra to generate fluorescence kinetic profiles as fluorescence emissions that can be detected in different channels.In such cases, each of the fluorescence kinetic profiles generated can be different, but using multiple channels means that a particular fluorescence kinetic profile can be replicated between sets.In other words, if two or more pairs that generate similar or even identical fluorescence kinetic profiles generate fluorescence kinetic profiles as fluorescence emissions that are observed in different channels, these two or more pairs that generate very similar or even identical fluorescence kinetic profiles can be used.

[0049] In this type of multiplexing, each T oligonucleotide still has a different sequence unique to the binding agent to which it is conjugated, and each different pair in the set generates a fluorescence kinetic profile that is unique to that pair within that set, but the use of FRET oligonucleotides that generate fluorescence kinetic profiles as fluorescence emissions that can be detected in different channels further enhances the multiplexing possibilities.

[0050] Illustrating the above, a given set of binder-T oligonucleotide conjugates may, for example, include 10 T oligonucleotides (T oligonucleotides 1-10). FRET oligonucleotide A (e.g., fluorescing "red") may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 1-10). For example, by adding an additional set of binder-T oligonucleotide conjugates including 10 T oligonucleotides (T oligonucleotides 11-20) and corresponding FRET oligonucleotides (FRET oligonucleotides B (e.g., also fluorescing "red")), 10 more different fluorescence kinetic profiles (profiles 11-20) may be used. Because FRET oligonucleotides A and FRET oligonucleotides B include emitter molecules with overlapping emission spectra (e.g., if they include the same emitter molecule), their fluorescence may be observed in the same channel (herein referred to as "red"), and therefore all 20 different fluorescence kinetic profiles may be observed in a single channel. Here, the binder-T oligonucleotide conjugates containing T oligonucleotides 1 to 10 are one set, and the binder-T oligonucleotide conjugates containing T oligonucleotides 11 to 20 are a second set, and thus the binder-T oligonucleotide conjugates containing T oligonucleotides 1 to 20 are a "set group."

[0051] A set of further binder-T oligonucleotide conjugates may be used, for example, the set of further binder-T oligonucleotide conjugates may include, for example, 10 T oligonucleotides (T oligonucleotides 21-30). FRET oligonucleotide C (e.g., emitting "green" fluorescence) may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 21-30). For example, a set of further binder-T oligonucleotide conjugates including 10 T oligonucleotides (T oligonucleotides 31-40) and a corresponding FRET oligonucleotide (FRET oligonucleotide D (e.g., also emitting "green" fluorescence)) may generate 10 more different fluorescence kinetic profiles (profiles 31-40). Because FRET oligonucleotide C and FRET oligonucleotide D include emitter molecules with overlapping emission spectra (e.g., they include the same emitter molecule), their fluorescence may be observed in the same channel (herein referred to as "green"), and thus all 20 different fluorescence kinetic profiles may be observed in a single channel. Here, the binder-T oligonucleotide conjugates containing T oligonucleotides 21 to 30 are one set, and the binder-T oligonucleotide conjugates containing T oligonucleotides 31 to 40 are a second set, and therefore the binder-T oligonucleotide conjugates containing T oligonucleotides 21 to 40 are a "set group."

[0052] Thus, in this example, these two sets (i.e., "multiple sets") can be used together in a multiplexed fluorescence microscopy method, where 40 binders can be used. Each binder-T oligonucleotide comprises a T oligonucleotide with a sequence that is unique to the binder to which it is conjugated. Furthermore, the dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is (i) unique to that pair within each set, and (ii) unique to that pair within each set. The dissociation and reassociation between each different pair can also generate a fluorescence kinetic profile that is unique to that pair within multiple sets, but this is not necessary, because two similar or identical fluorescence kinetic profiles can be used when the two fluorescence kinetic profiles are generated by FRET oligonucleotides with different emitter molecules (e.g., emitter molecules with emission spectra, such as different fluorescence emission spectra, that can be observed in different channels).

[0053] Thus, in the theoretical example above, oligonucleotides can be designed to generate 40 different fluorescence kinetic profiles. For example, if pairs using FRET oligonucleotide A and one T oligonucleotide, and FRET oligonucleotide C and another T oligonucleotide both produce similar or identical fluorescence kinetic profiles, oligonucleotides that generate fewer than 40 different fluorescence kinetic profiles can be used. This is because the fluorescence kinetic profiles obtained using FRET oligonucleotide A and those obtained using FRET oligonucleotide C are detected in different channels.

[0054] Thus, the above shows how further multiplexing can be achieved when using the present invention.It is not necessary to construct multiplexing exactly as above.For example, it is possible to use multiple sets of binder-T oligonucleotide conjugates, where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in the channel specific to that set, and the step of observing the fluorescence kinetic profile of sample includes observing the fluorescence kinetic profile of sample in the channel for each set.

[0055] For example, a first set of binder-T oligonucleotide conjugates may include, for example, 10 T oligonucleotides (T oligonucleotides 1-10). FRET oligonucleotide A (red) may, for example, reversibly hybridize to these 10 T oligonucleotides to generate 10 different fluorescence kinetic profiles (profiles 1-10). A second set of binder-T oligonucleotide conjugates, for example, including 10 T oligonucleotides (T oligonucleotides 11-20) and the corresponding FRET oligonucleotide (FRET oligonucleotide B (green)), generates 10 different fluorescence kinetic profiles (profiles 11-20). Each binder-T oligonucleotide includes a T oligonucleotide with a sequence unique to the binder to which it is conjugated. Dissociation and reassociation between each different pair generates a fluorescence kinetic profile unique to that pair within each set. A fluorescence kinetic profile may be, but is not necessarily, unique to that pair within multiple sets. Dissociation and reassociation between each different pair may also generate a fluorescence kinetic profile that is unique to that pair within a set, although this is not required as two similar or identical fluorescence kinetic profiles may be used if the two fluorescence kinetic profiles are generated by FRET oligonucleotides with different emitter molecules (e.g., emitter molecules having emission spectra, such as fluorescence emission spectra, that can be observed in different channels).

[0056] In another example, one or more sets of binder-T oligonucleotide conjugates, in which the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set, are used together with one or more sets of binder-T oligonucleotide conjugates, in which the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set.In other words, a (for example, one) set of binder-T oligonucleotide conjugates can be used together with a (for example, one) set of binder-T oligonucleotide conjugates (where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set). A (for example, one) set of binder-T oligonucleotide conjugates may be used with a plurality of sets of binder-T oligonucleotide conjugates (wherein the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set). A plurality of sets of binder-T oligonucleotide conjugates, where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set, may be used with a (for example, one) set of binder-T oligonucleotide conjugates, where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to the set.A plurality of sets of binder-T oligonucleotide conjugates, in which the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to that set, may be used together with a plurality of sets of binder-T oligonucleotide conjugates, in which the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to that set.

[0057] Specific examples of how multiplexing can be achieved in accordance with the present invention include those summarized in Table 1. [Table 1-1] [Table 1-2]

[0058] sample A sample may generally comprise one or more cells (e.g., a population of cells) or may comprise a cell lysate from a cell or a population of cells. A sample may comprise prokaryotic or eukaryotic cells, such as animal, plant, yeast, bacterial, or other cells. The cells are preferably animal cells. They may be of animal origin, particularly mammalian origin. They may be primary cells or cells that are cell lines, such as mammalian cell lines. A sample may comprise cells from an animal model of a disease or a human or animal patient with a disease. A sample may be a biological sample, for example, from an animal (preferably a mammal, such as a human or an experimental animal, such as a mouse or rat), and the sample may be, for example, blood, sputum, lymph, mucus, feces, urine, etc. A sample may be a tissue sample, such as a tissue section. A sample may be an environmental sample, such as a water sample, an air sample, a food sample, etc.

[0059] Fixation of samples The method of the present invention is carried out on a fixed sample, which may be a chemically fixed sample. Fixation of samples for fluorescence microscopy is well known and is carried out to preserve the sample (e.g., to maintain the morphology of cells in a structurally close state to their living state). When using excised tissue, fixation can also prevent its autolysis and necrosis. It can also preserve antigenicity and allow the components used in the method to more easily access the interior parts of the cells.

[0060] The method of the present invention may further comprise one or more steps of fixing the sample, for example to preserve the sample. This may be performed before step a) of the method described herein. After the fixed sample is contacted with a set of binder-T oligonucleotide conjugates to bind the binder with any binding partner present in the sample, and before the sample and any binder bound as a result of step a) are contacted with the FRET oligonucleotide, a further optional fixation step may be performed. The further fixation step at this stage of the method is advantageous to further immobilize the bound binder on the sample. Although the binder may be bound to its target molecule, for example using electrostatic binding, fixation may improve further processing steps of the sample. This improvement may occur as a result of fixation creating a stronger bond between the bound binder and the target molecule in the sample, for example covalently crosslinking the bound binder and the target molecule in the sample.

[0061] Fixation, such as chemical fixation, is well known in the art.

[0062] Examples of suitable methods for carrying out chemical fixation include the use of cross-linking fixatives, such as those based on aldehydes (e.g., to create covalent chemical bonds between proteins in a sample), or oxidative cross-linking fixatives. Examples of aldehyde-based fixatives include formaldehyde, glutaraldehyde, glyoxal, and paraformaldehyde. Oxidative cross-linking fixatives are, for example, osmium tetroxide, chromate, potassium dichromate, and potassium permanganate. Alternatively, precipitation fixatives may be used, such as acetone, ethanol, and methanol. Alternative agents include Hepes-glutamate buffer-mediated organic solvent protective effect (HOPE) fixative, mercurials such as B-5 and Zenker fixative, and picrate salts. Preferably, aldehyde cross-linking fixatives are used.

[0063] Permeabilization of samples Permeabilization of the sample may be performed. Thus, in some embodiments, the method includes one or more steps of permeabilizing the sample, for example to allow the molecules used in the method to access the interior of the cells. This can be performed before step a) of the method described herein and before, after, or simultaneously with any fixation step. Suitable permeabilization agents and protocols are known in the art and include the use of agents such as saponin, Triton-X, and Tween-20. Suitable protocols are, for example, described in Jamur MC, Oliver C. ((2010) Permeabilization of Cell Membranes. In: Oliver C., Jamur M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology (Methods and Protocols), vol 588. Humana Press) or as described in https: / / www.abcam.com / ps / pdf / protocols / fixation_permeabilization.pdf.

[0064] Further sample processing In step a), the fixed sample is contacted with a set of binder-T oligonucleotide conjugates to allow the binder to bind to any binding partners present in the sample. Typically, the sample is fixed and thus attached to a solid support at any suitable time for fluorescence microscopy. The contacting step can be carried out in any suitable manner that allows the set of binder-T oligonucleotide conjugates to bind to any binding partners present in the sample. This is typically by applying a solution containing the set of binder-T oligonucleotide conjugates to the solid support to which the sample is attached. The binder-T oligonucleotide conjugates can be present in the solution at a concentration of, for example, 5-50ug / ml (or 1-500, 2-250ug / ml).

[0065] Suitable conditions include, for example, incubation in a buffer containing a binder-T oligonucleotide conjugate and a blocking reagent, such as BSA or salmon sperm DNA, at about 4°C, or at about 15-25°C.

[0066] The contacting can continue for any suitable length of time to allow the set of binder-T oligonucleotide conjugates to bind to any binding partners. Exemplary periods include 10 minutes to 2 hours, for example 30 minutes to 1 hour.

[0067] The advantage of the method described herein is that no washing step is required and the background fluorescence level is generally not high, for example compared to the DNA-PAINT method described above. This means that a washing step to remove unbound components is generally not required to obtain good quality data. However, after step a, a step of removing unbound binder-T oligonucleotide conjugates may be performed. This may be performed to further reduce the background fluorescence level, since FRET oligonucleotides may bind to unbound binder-T oligonucleotide conjugates, which may cause background fluorescence.

[0068] A washing step can be performed, for example, by removing the solution containing the binder-T oligonucleotide conjugate and replacing it with a liquid that does not contain the binder-T oligonucleotide conjugate, for example one or more changes of a suitable buffer.

[0069] The step of contacting the sample and any binding agents bound as a result of step a with the FRET oligonucleotide may be performed immediately after step a or after one or more washing steps. The contacting step may be performed in any suitable manner that allows the FRET oligonucleotide to hybridize to one or more T oligonucleotides. In this way, multiple pairs are formed. This is generally done by applying a solution containing the FRET oligonucleotide to the sample and any binding agents bound as a result of step a. The FRET oligonucleotide may be present in the solution at a concentration of, for example, 1 pM to 1 uM (or 10 pM to 500 nM, 20 pM to 250 nM, 50 pM to 100 nM, 100 pM to 50 nM, 200 pM to 25 nM, 500 pM to 1 nM).

[0070] The steps of illuminating the sample with a wavelength that causes excitation of the emitter molecule and observing the kinetic fluorescence profile of the sample over time at one or more pixels generally occur after the sample is in contact with the FRET oligonucleotide. Thus, the illumination and observation steps occur while the sample is in contact with the FRET oligonucleotide and may continue for as long as the sample is in contact with the FRET oligonucleotide or for a suitable period of time to capture and optionally record the fluorescence kinetic profile.

[0071] Oligonucleotides Oligonucleotides, such as T oligonucleotides or FRET oligonucleotides, are linear polymeric molecules that contain linked units, which units contain nucleobases or functional equivalents thereof. Nucleobases can be naturally occurring nucleobases, such as adenine, thymine, guanine, cytosine, uracil, inosine, pseudouridine, dihydrouridine, 7-methylguanosine (preferably selected from adenine, thymine, guanine, cytosine, and uracil), and derivatives thereof, or non-naturally occurring nucleobases, such as isocytosine, isoguanine. The unit that contains a nucleobase can be a nucleoside (nucleobase and a sugar group, such as a pentose sugar, preferably ribose or deoxyribose), but is preferably a nucleotide (nucleoside and phosphate). Nucleotides are the monomeric units of DNA and RNA, and the oligonucleotides used in the methods of the present invention preferably contain nucleotides, such as deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0072] Preferably, the oligonucleotide does not include fluorescent nucleobases.In a preferred embodiment, the oligonucleotide is or comprises DNA.

[0073] Well-known examples of oligonucleotides include those that are or contain DNA, RNA, or other nucleic acid-like structures. They can have a phosphate-sugar backbone or a backbone that contains non-phosphate-sugar moieties (e.g., peptide nucleic acid (PNA) and morpholino). Phosphate-sugar backbone analogs include PNA, morpholino, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA), and hexitol nucleic acid (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

[0074] The present invention utilizes the ability of oligonucleotides as single-stranded molecules to hybridize with complementary or partially complementary oligonucleotides to form double-stranded pairs.Two fully complementary or partially complementary oligonucleotides can bind to each other to form double-stranded molecules based on complementary base pairing, as is well known in the art.

[0075] In the method of the present invention, the T oligonucleotides and their corresponding FRET oligonucleotides are designed to have sequences that are generally only partially complementary, so that the oligonucleotide pairs formed between the T oligonucleotides and their corresponding FRET oligonucleotides are only temporarily in double-stranded form. Each pair undergoes a process of association and dissociation over a period of time. Because the FRET oligonucleotides contain a fluorescent emitter molecule and a quencher, the transient nature of the hybridization is reflected in the fluorescence kinetic profile. When the FRET oligonucleotide is in solution, the proximity of the quencher to the fluorescent emitter molecule means that when irradiated with light of the appropriate wavelength, the fluorescence emitted from the emitter molecule is quenched by the quencher and cannot be detected at the emission wavelength of the emitter molecule. When the FRET oligonucleotide hybridizes to the T oligonucleotide, it constrains the structure of the FRET oligonucleotide, physically separating the quencher and the fluorescent emitter molecule, so that when irradiated with light of the appropriate wavelength, the fluorescence emitted from the emitter molecule is not quenched by the quencher and can be observed at the emission wavelength of the emitter molecule. Thus, association between the two members of each pair is associated with fluorescence at the emission wavelength of the emitter molecule, and dissociation is associated with an absence of fluorescence at the emission wavelength of the emitter molecule.

[0076] In each set of T oligonucleotides, the members of the set have different sequences and dissociate and reassociate with the corresponding FRET oligonucleotide in a manner that depends on their sequences. Thus, the hybridization kinetics of the two oligonucleotides of each pair determine the fluorescence kinetic profile that can be observed.

[0077] When oligonucleotide is not bound to its complementary or partially complementary pair, it may be single-stranded and may not have stable secondary structure.In a preferred embodiment, the oligonucleotide used herein does not form stable secondary structure such as hairpin loop in single-stranded form.This provides an advantage in the present invention.Because the oligonucleotide that does not form such stable secondary structure can form pair more easily and therefore more quickly, and is more suitable for imaging.

[0078] T oligonucleotide The T oligonucleotide has a sequence specific to the binding agent to which it is conjugated. Also, the T oligonucleotide can hybridize to its corresponding FRET oligonucleotide. The sequence of an oligonucleotide is determined by the nature and order of the units that compose it. Standard terms can be used to define the sequence (e.g., with respect to the base sequence of the molecule).

[0079] T oligonucleotides are generally 8-35 nucleotides or equivalent units in length, where one nucleotide unit is the size of a standard DNA nucleotide unit. Alternatively, T oligonucleotides can be 9-30, 10-29, 11-28, 12-27, 13-26, 14-25, 15-24, 16-23, 17-22, 18-21, or 10-19, 11-18, 12-17, 13-18, 14-17, 15-16 nucleotide units in length (e.g., up to 35, 34, 33, 32, 31, 30, 29, 28, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 nucleotide units in length).

[0080] A T oligonucleotide is generally capable of hybridizing to its corresponding FRET oligonucleotide and generally does not hybridize to other T oligonucleotides in the set, or other T oligonucleotides in the set group, if used. Generally, each T oligonucleotide hybridizes to its corresponding FRET oligonucleotide and does not hybridize to FRET oligonucleotides used in the method other than its corresponding FRET oligonucleotide.

[0081] The T oligonucleotide and its corresponding FRET oligonucleotide contain complementary nucleotides or their functional equivalents. When complementary bases are present in the two oligonucleotide strands, hydrogen bonds can occur between the complementary bases. Hybridization is a non-covalent, sequence-specific interaction between two oligonucleotide strands, forming a single complex of the two strands.

[0082] Two complementary strands with one or more mismatches can also bind to each other, but do so less readily because the mismatches make binding between them less energetically favorable. Changes to the binding characteristics depend at least on (i) the length of the oligonucleotide (e.g., over the overlap region), (ii) the sequence composition, (ii) the degree of complementarity (e.g., over the overlap region), (iii) the location of the mismatch in the two strands (adjacent mismatches generally have a greater effect on the stability of the interaction between the two strands than nonadjacent mismatches), and (iv) the nature of the complementary pair (e.g., a pair of nucleobases A=T forms a less stable bond than a G≡C pair).

[0083] As described elsewhere herein, the T oligonucleotides are generally 8-35 or 9-20 oligonucleotide units in length, and the FRET oligonucleotides are (i) longer than the Förster radius of the FRET oligonucleotide emitter molecule, and / or (ii) at least 12 nucleotide units in length.

[0084] In general, the strength with which two oligonucleotides hybridize also depends on the degree of complementarity between the two strands (i.e., the number of bases that are complementary relative to the total length of each oligonucleotide, or relative to the length of overlap between the two oligonucleotides if the sequences are not the same length). A certain degree of sequence complementarity is necessary for hybridization to occur.

[0085] Typically, there is a mismatch between the T oligonucleotide and its corresponding FRET oligonucleotide in the range of 0-40%, 10-40%, or 20-40% compared to the maximum number of base pairs that can be formed in such a pair. The T oligonucleotide is typically 8-35 nucleotide units in length, and the corresponding FRET oligonucleotide is typically longer than the Förster radius of the FRET oligonucleotide emitter molecule, and / or (ii) at least 12 nucleotide units in length. The percentage of mismatch is defined as the percentage of mismatch compared to the maximum number of base pairs that can be formed, taking into account that the two oligonucleotides are not always the same length. In other words, if the T oligonucleotide is 10 nucleotide units long and the corresponding FRET oligonucleotide is 15 nucleotide units long, the maximum number of base pairs that can be formed in the pair is 10, and therefore a 0-40% mismatch in the pair corresponds to 0-4 mismatches in the overlapping 10 nucleotide unit section, and a 20-40% mismatch in the pair corresponds to 2-4 mismatches in the overlapping 10 nucleotide unit section. This is shown in the table below, where for each pair, an exemplary number of mismatches that result in a 20-40% mismatch is shown along with the maximum number of base pairs that can be formed in each pair (shown in brackets). In another example, where the T oligonucleotide is 12 nucleotide units long and the FRET oligonucleotide is also 12 nucleotide units long, the maximum number of base pairs that can be formed in the pair is 12, and the number of mismatches that correspond to this 20-40% is 2-4.

[0086] The number of mismatches corresponding to 20-40% for certain preferred combinations of T oligonucleotides and FRET oligonucleotides is shown in Table 2 below. [Table 2]

[0087] Thus, generally, a T oligonucleotide has 1-8, 2-7, 3-6 mismatches, such as 1-3, 2-4, 2-5, 3-6, 4-8 mismatches with its corresponding FRET oligonucleotide.

[0088] If the maximum number of base pairs that can be formed in a pair is 9 (e.g., if the T oligonucleotide is 9 nucleotide units long and the FRET oligonucleotide is at least 12 nucleotide units long), then there can be 1 to 3 mismatches.

[0089] If the maximum number of base pairs that can be formed in a pair is 10 (e.g., if the T oligonucleotide is 10 nucleotide units long and the FRET oligonucleotide is at least 12 nucleotide units long), then there can be 2 to 4 mismatches.

[0090] If the maximum number of base pairs that can be formed in a pair is 11 (e.g., if the T oligonucleotide is 11 nucleotide units long and the FRET oligonucleotide is at least 12 nucleotide units long), then there can be 2 to 4 mismatches.

[0091] If the maximum number of base pairs that can be formed in a pair is 12 (e.g., if the T oligonucleotide is 12 nucleotide units long and the FRET oligonucleotide is at least 12 nucleotide units long, or if the T oligonucleotide is 13-20 nucleotide units long and the FRET oligonucleotide is 12 nucleotide units long), then there can be 2-4 mismatches.

[0092] If the maximum number of base pairs that can be formed in a pair is 13 (e.g., if the T oligonucleotide is 13 nucleotide units long and the FRET oligonucleotide is at least 13 nucleotide units long, or if the T oligonucleotide is 14-20 nucleotide units long and the FRET oligonucleotide is 13 nucleotide units long), then there can be 2-5 mismatches.

[0093] If the maximum number of base pairs that can be formed in a pair is 14 (e.g., if the T oligonucleotide is 14 nucleotide units long and the FRET oligonucleotide is at least 14 nucleotide units long, or if the T oligonucleotide is 15-20 nucleotide units long and the FRET oligonucleotide is 14 nucleotide units long), then there can be 2-5 mismatches.

[0094] If the maximum number of base pairs that can be formed in a pair is 15 (e.g., if the T oligonucleotide is 15 nucleotide units long and the FRET oligonucleotide is at least 15 nucleotide units long, or if the T oligonucleotide is 16-20 nucleotide units long and the FRET oligonucleotide is 15 nucleotide units long), then there can be 3-6 mismatches.

[0095] If the maximum number of base pairs that can be formed in a pair is 16 (e.g., if the T oligonucleotide is 16 nucleotide units long and the FRET oligonucleotide is at least 16 nucleotide units long, or if the T oligonucleotide is 17-20 nucleotide units long and the FRET oligonucleotide is 16 nucleotide units long), then there can be 3-6 mismatches.

[0096] If the maximum number of base pairs that can be formed in a pair is 17 (e.g., if the T oligonucleotide is 17 nucleotide units long and the FRET oligonucleotide is at least 17 nucleotide units long, or if the T oligonucleotide is 18-20 nucleotide units long and the FRET oligonucleotide is 17 nucleotide units long), then there can be 3-6 mismatches.

[0097] If the maximum number of base pairs that can be formed in a pair is 18 (e.g., if the T oligonucleotide is 18 nucleotide units long and the FRET oligonucleotide is at least 18 nucleotide units long, or if the T oligonucleotide is 19-20 nucleotide units long and the FRET oligonucleotide is 18 nucleotide units long), then there can be 3-7 mismatches.

[0098] If the maximum number of base pairs that can be formed in a pair is 19 (e.g., if the T oligonucleotide is 19 nucleotide units long and the FRET oligonucleotide is at least 19 nucleotide units long, or if the T oligonucleotide is 20 nucleotide units long and the FRET oligonucleotide is 19 nucleotide units long), then there can be 3 to 7 mismatches.

[0099] If the maximum number of base pairs that can be formed in a pair is 20 (e.g., if the T oligonucleotide is 20 nucleotide units long and the FRET oligonucleotide is at least 20 nucleotide units long), then there can be 4 to 8 mismatches.

[0100] Mismatches can occur where no base pair occurs between the bases on the two strands. This may be due to the absence of a complementary base at the appropriate position in the T oligonucleotide or FRET oligonucleotide. Non-complementary base pairs can include or consist of naturally occurring bases (e.g., A, T, C, G, U). Examples of naturally occurring non-complementary base pairs include AG, GA, AC, CA, AA, TT, TG, GT, TC, CT, CC, GG, UU, UG, GU, UC, CU pairs. The presence of other bases (e.g., non-naturally occurring or modified bases) can also cause mismatches.

[0101] Position (or relative position when there are multiple mismatches) also contributes to the stability of oligonucleotide pairs. The presence of two or more adjacent mismatches tends to affect hybridization to a greater extent than two mismatches that are not adjacent (e.g., separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 complementary base pairs). Thus, the T oligonucleotide used according to the present invention can have a sequence that creates two or more adjacent mismatches, such as three or more, or four or more, or five or more adjacent mismatches. Thus, other T oligonucleotides used according to the present invention can have a sequence that creates two mismatches that are not adjacent (e.g., separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 complementary base pairs), or at least three mismatches that are not adjacent (e.g., all pairs of mismatches are separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 complementary base pairs). Additional T oligonucleotides used in accordance with the invention may have sequences that give rise to a mixture of adjacent and non-adjacent mismatches.

[0102] Hybridization is further hindered when the mismatch includes G or C bases at the end of T oligonucleotide, for example within 5, 4, 3, 2 nucleotide units of the end of T oligonucleotide.Therefore, the T oligonucleotide used according to the present invention can have a sequence that causes one or more mismatches located at the end of T oligonucleotide, for example within 5, 4, 3, 2 nucleotide units of the end of T oligonucleotide.Preferably, one of the mismatch bases in each mismatch pair is G or C or its functional equivalent (for example, G or C can be present in T oligonucleotide or corresponding FRET oligonucleotide at this position where mismatch exists).

[0103] Including mismatches that create flexible overhangs or generate new structural conformations further hinders hybridization. The introduction of mismatches can also generate mononucleotide repeats. Mononucleotide repeats are single nucleotide repeats, and therefore, for example, one or more mismatches (e.g., when the repeat is at least 2, 3, 4, 5, 6 nucleotides) can be used, where at least one nucleotide in the mismatch is part of the mononucleotide repeat. The introduction of mismatches can also generate secondary structures that can affect hybridization between FRET oligonucleotides and T oligonucleotides (e.g., the formation of hairpin structures in T oligonucleotides that are at least 2, 3, 4, 5 nucleotide units long).

[0104] In view of the above, any individual T oligonucleotide used in accordance with the present invention may have one or more of the above characteristics: (i) 0-40%, 10-40%, or 20-40% mismatch with its corresponding FRET oligonucleotide compared to the maximum number of base pairs that can be formed in such pair; (ii) two or more adjacent mismatches (e.g., three or more, or four or more, or five or more adjacent mismatches); (iii) two or more non-contiguous mismatches (e.g., separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 complementary base pairs, or, if there are more than two non-contiguous mismatches, each pair of mismatches is separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 complementary base pairs); (iv) one or more mismatches located within 5, 4, 3, 2 nucleotide units of the end of the T oligonucleotide (optionally, one of the mismatched bases in each mismatch pair is G or C or its functional equivalent (e.g., G or C can be present in the T oligonucleotide or the corresponding FRET oligonucleotide at this position such that there is a mismatch)); (v) one or more mismatches that create a flexible overhang; (vi) one or more mismatches that generate a new structural conformation; (vii) one or more mismatches where at least one nucleotide in the mismatch is part of a mononucleotide repeat (e.g., where the repeat is at least 2, 3, 4, 5, 6 nucleotides); (viii) One or more mismatches in which at least one nucleotide in the mismatch forms part of a secondary structure that may affect hybridization between the FRET oligonucleotide and the T oligonucleotide (e.g., the formation of a hairpin structure in the T oligonucleotide of at least 2, 3, 4, 5 nucleotide units in length).

[0105] Set of binder-T oligonucleotide conjugates In the method according to the present invention, a set of binder-T oligonucleotide conjugates is used.In the set, a plurality of binder-T oligonucleotide conjugates are used.Since the method is based on the ability to generate different fluorescence kinetic profiles, each of which is due to the presence of a specific T oligonucleotide, the selection of the different T oligonucleotides that compose each set must be performed in such a way that a plurality of fluorescence kinetic profiles are generated.

[0106] Each set of T oligonucleotides has a corresponding FRET oligonucleotide. The T oligonucleotides in each set are selected so that the association and dissociation of each T oligonucleotide and its corresponding FRET oligonucleotide results in different fluorescence kinetic profiles. Each set of binder-T oligonucleotide conjugates includes two or more different T oligonucleotides, and the structural features (e.g., sequence) of the T oligonucleotides determine the fluorescence kinetic profile of the pair. Thus, the set includes T oligonucleotides that generate different fluorescence kinetic profiles as a result of association and dissociation with the corresponding FRET oligonucleotide.

[0107] For example, for a given FRET oligonucleotide, one member of a set of T oligonucleotides may produce a pair that associates and dissociates relatively quickly, for example, at a frequency of 15 blinking events per second, and another member of a set of T oligonucleotides may produce a pair that associates and dissociates more slowly, for example, at a frequency of 5 blinking events per second.In general, an oligonucleotide pair with a large number of mismatches will have a higher frequency of blinking events than a pair with a small number of mismatches.In general, when comparing two pairs with completely complementary overlap regions, a pair with a longer overlap region will have a longer off time and a longer on time than a pair with a shorter overlap region.

[0108] As mentioned elsewhere herein, a set as defined herein is one that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or at least ... , 25, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, such as 1-25, 2-24, 3-23, 4-22, 5-21, 6-20, 7-19, 8-18, 9-17, 10-16, 11-15, 12-14 binding agent-T oligonucleotide conjugates.

[0109] A T oligonucleotide generally hybridizes only to its corresponding FRET oligonucleotide, and not to other FRET oligonucleotides or other T oligonucleotides.

[0110] A preferred set of T oligonucleotides and corresponding FRET oligonucleotides include: - A set containing (or possibly consisting of) the following: o SEQ ID NO:1 (5'-TTCCACATTACTTCT-3') and SEQ ID NO: 2 (5'-ATCCCCATTACTTCT-3') one or both of the T oligonucleotides of the sequence And o the corresponding FRET oligonucleotide of sequence SEQ ID NO: 7 (5'-AGAAGTAATGTGGAA-3'), - A set containing (or possibly consisting of) the following: o SEQ ID NO: 3 (5'-GGGTGGCGTATGATAGCTAT-3'), SEQ ID NO: 4 (5'-AGGTTAAGTATGATAGTTAT-3'); SEQ ID NO:5 (5'-GGGTTAAGCATGGTAGTTAT-3') and SEQ ID NO:6 (5'-AGGTTAAGCGTTATATTTAT-3') at least one of the T oligonucleotide(s) of the sequence (e.g., at least two, three, or all four of the listed T oligonucleotides); And o The corresponding FRET oligonucleotide of sequence SEQ ID NO: 8 (5'-ATAACTATCATACTTAACCT-3').

[0111] The above two sets can be used together as a set, for example, when the two FRET oligonucleotides contain emitter molecules with overlapping emission spectra, and optionally have the same emitter molecule.

[0112] Binding Agents and Targeting Molecules A binder is any agent that can bind to a binding partner or a target molecule of interest, e.g., directly or indirectly binds to the target molecule of interest. A binder is generally specific to the target molecule of interest, e.g., can bind to the target molecule of interest with higher affinity and / or specificity than to other components in the sample. A binder generally binds to the target molecule of interest directly, e.g., via electrostatic binding.

[0113] A target molecule can be any molecule or entity of interest that requires detection. For example, a target molecule can include peptides or proteins, nucleic acids (RNA, DNA, or any modified form thereof), lipids, carbohydrates, small molecules. A target molecule can be present in or on a cell. Since the method of the present invention allows for the study of multiple target molecules, it is not necessary that all target molecules are of the same type. Thus, the method can result in the observation of one or more of peptides or proteins, nucleic acids (RNA, DNA, or any modified form thereof), lipids, carbohydrates, or small molecules, or a combination of some or all of these.

[0114] The binding agent may be or include a protein (e.g., a polypeptide) or may be or include a non-protein molecule. Examples of binding agents that are or include proteins include antibodies, antibody fragments, receptors (e.g., soluble receptors), ligands, and lectins. Examples of binding agents that are or include non-protein molecules include carbohydrates and nucleic acid molecules or their derivatives, as well as aptamers.

[0115] A binding agent has a high binding affinity for its target, e.g., a dissociation constant in the micromolar or nanomolar range (e.g., about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 Less than M or 10 -4 M~10 -6 M or 10 -7 M~10 -9 The affinity of the antibody may be selected to have a binding affinity in the range of 1 μM (M range). Affinity is the strength with which a molecule (e.g., an antibody) binds to its ligand (e.g., an antigen). It is usually measured and reported by the equilibrium dissociation constant (KD). KD is the ratio of the antibody association rate ("on rate" or Kon), i.e., how fast the antibody binds to its antigen, to the antibody dissociation rate ("off rate" or Koff), i.e., how fast the antibody dissociates from its antigen. For example, an antibody with an affinity of 1 μM or less has a KD value of 1 μM or less (i.e., affinity of 1 μM or more), as determined by a suitable in vitro binding assay. Suitable in vitro assays, such as biolayer interferometry (e.g., Octet) or surface plasmon resonance (e.g., Biacore System), can be used to assess affinity as measured by KD values ​​based on well-known methods. Selecting a binder with high binding affinity for a target molecule can increase the selectivity and sensitivity of the method of the invention. The term "antibody" refers to an antibody having at least one V H and / or V LIt is used to mean an immunoglobulin protein that contains domains and binds to a target molecule. Many antibody structures are known, including full-length antibodies, which contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and heavy-chain-only antibodies such as HCAbs of camelids and sharks. An antibody fragment may include an antibody or an antigen-binding portion thereof. Antibodies may be polyclonal or monoclonal, naturally occurring or synthetic, or modified forms thereof (e.g., humanized, chimeric).

[0116] The antigen-binding function of an antibody may be performed by a fragment of a full-length antibody. As used herein, an "antigen-binding portion" of an antibody, e.g., a monoclonal antibody, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment (a monovalent fragment containing the VH, VL, CL and CH1 domains), (ii) a F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region), (iii) a Fd fragment consisting of the VH and CH1 domains, (iv) a Fv fragment (the VH and VL domains of one arm of an antibody (including scFv fragments, e.g., fragments in which the VH and VL portions are joined by a synthetic linker, as well as multimers thereof)), (v) a dAb fragment (a single VH domain), and (vi) an isolated complementarity determining region (CDR), or a combination of at least two CDRs, which may be joined, e.g., by a synthetic linker.

[0117] These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0118] Examples of binding agents that are or comprise non-protein molecules include binding agents that comprise oligonucleotides (which may be, for example, DNA, RNA or synthetic oligonucleotides, which may comprise deoxyribonucleotide, ribonucleotide and / or synthetic nucleotide residues).

[0119] Conjugation of the binding agent and the T oligonucleotide The binding agent and the T oligonucleotide are conjugated. This means that these two components of the conjugate are linked by covalent or non-covalent bonds. This can be achieved by any suitable means. The components of the conjugate can be linked directly or indirectly, for example via a linker.

[0120] In embodiments where the binder is a nucleic acid or an aptamer, the coupling can be, for example, via a covalent phosphodiester bond between the 5' phosphate group of one nucleotide or functional equivalent thereof and the 3' OH group of another nucleotide or functional equivalent thereof. In such embodiments, the binder component and the T oligonucleotide component can be matched or separated by one or more oligonucleotide units. In such cases, the oligonucleotide unit that is not involved in binding to the binding target or FRET oligonucleotide can be referred to as a linked oligonucleotide unit. For example, there can be 2-30, 3-25, 4-20, 5-15, 6-10 linked oligonucleotide units, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10 linked oligonucleotide units.

[0121] In embodiments where the binding agent is a protein such as an antibody, conjugation between the binding agent and the T oligonucleotide can be by any suitable covalent or non-covalent linkage. Suitable means known in the art include chemical cross-linking of the two components of the conjugate, for example, via a linker such as a cross-linking agent. Cross-linking reagents contain reactive ends for specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. The availability of several chemical groups on proteins and peptides makes them targets for conjugation using cross-linking methods. Cross-linking agents contain at least two reactive groups that target common functional groups found on biomolecules such as proteins and nucleic acids. Functional groups commonly targeted for bioconjugation include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids. Other examples include click chemistry using modified (e.g., maleimide, NHS, or azide groups) T oligos. The two reactive groups of the cross-linking agent are separated by a spacer that may include, for example, PEG. The spacer is generally between 3 and 50 angstroms in length.

[0122] The T Oligo is preferably conjugated at a location on the binding agent that does not interfere with the ability of the binding partner to bind to its binding partner, e.g., in the case of a binding agent that is an antibody or antibody fragment, the T Oligo is preferably conjugated at the constant region and / or in a region that does not interfere with the antibody binding to its target, e.g., away from the antigen binding site.

[0123] Each molecule of the binding agent can be conjugated to a single T oligonucleotide molecule, or to multiple T oligonucleotide molecules. When multiple T oligonucleotide molecules are conjugated to each molecule of the binding agent, each of these T oligonucleotide molecules has the same oligonucleotide sequence (because the sequence of the T oligonucleotide is unique to the binding agent to which it is conjugated).

[0124] For example, 1-10, 2-9, 3-8, 4-7, or 5-6 T oligonucleotide molecules are conjugated to each molecule of the binding agent. Each T oligonucleotide molecule may be conjugated at a different position on the binding agent. The number of T oligonucleotide molecules conjugated to each binding agent molecule depends on the nature of the conjugation used. This number of molecules of T oligonucleotide per molecule of the binding agent can be absolute, for example, when each molecule of the binding agent has the same number of molecules of T oligonucleotide. Alternatively, the number of molecules of T oligonucleotide per molecule of the binding agent can represent the average number of T oligonucleotides per molecule of the binding agent.

[0125] FRET oligos A FRET oligonucleotide is an oligonucleotide as defined elsewhere herein. A fluorescent emitter molecule (emitter molecule) and a quencher are bound, incorporated and / or conjugated to the oligonucleotide. The fluorescent emitter molecule may be a Förster resonance energy transfer (FRET) donor, and the quencher may be a corresponding FRET acceptor. When the FRET oligonucleotide is in solution and irradiated with a wavelength that causes excitation of the emitter molecule, no fluorescence is observed at the emission wavelength of the emitter molecule due to efficient energy transfer to the quencher. However, hybridization of the FRET oligonucleotide with a T oligonucleotide physically separates the emitter and quencher, slowing down the rate of energy transfer, e.g., FRET, between the emitter and quencher, so that fluorescence can be observed at the emission wavelength of the emitter molecule.

[0126] Thus, the emitter molecule and quencher are positioned on the oligonucleotide such that when the FRET oligonucleotide is fully linear (e.g., when the FRET oligonucleotide hybridizes to a T oligonucleotide or an oligonucleotide that is fully complementary in sequence to the FRET oligonucleotide), the emitter molecule and quencher are spatially separated, such that no energy transfer occurs between the two molecules and fluorescence can be observed at the emission wavelength of the emitter molecule.

[0127] In contrast, when the FRET oligonucleotide is in solution, its configuration is disordered and the fluorescent emitter molecule and the quencher are not spatially separated. Therefore, no energy transfer occurs between the emitter molecule and the quencher, and no or no fluorescence can be observed at the emission wavelength of the emitter molecule. Depending on the nature of the quencher, the energy transfer can result in quenching or suppression of all fluorescence (e.g., when a dark quencher or black hole quencher is used, because dark quenchers generally absorb the excitation energy from the emitter molecule and dissipate the energy as heat, i.e., no fluorescence is detected at all). Alternatively, the quencher molecule itself may emit fluorescence, which is emitted from the quencher molecule at a wavelength different from the emission wavelength of the emitter molecule. This means that, regardless of the nature of the quencher or the mechanism of quenching, quenching of the emission of the emitter molecule reduces or eliminates the fluorescence emission at the emission wavelength of the emitter molecule. The reduction can be to less than 60, 50, 40, 30, 20, 10% of the fluorescence emission observed at the emission wavelength of the emitter molecule in the absence of the quencher entity.

[0128] Considering the above, broadly speaking, fluorescence emission is detectable from the fluorescent emitter molecule in the FRET oligonucleotide at the emission wavelength of the emitter molecule when the FRET oligonucleotide is hybridized to the T oligonucleotide, but is not detected (or is detected at a reduced level) when the FRET oligonucleotide is free in solution. When the FRET oligonucleotide and the T oligonucleotide are associated, the fluorescence emission at the emission wavelength of the emitter molecule is "on", and when dissociated, the fluorescence emission at the emission wavelength of the emitter molecule is "off". Because the FRET oligonucleotide and the T oligonucleotide are designed to be incompletely hybridized, they easily dissociate and reassociate, giving rise to a unique fluorescence kinetic profile characteristic of each pair. The fluorescence kinetic profile is composed of both the frequency of blinking and the duration of the "on" signal, as described in more detail below.

[0129] Thus, the fluorescent emitter molecule and the quencher are attached, incorporated and / or conjugated to the oligonucleotide at a distance that prevents energy transfer (e.g., FRET) between the fluorescent emitter molecule and the quencher when the oligonucleotide is in a bound conformation. In other words, the distance between the two components in the oligonucleotide exceeds the Förster radius of the FRET oligonucleotide emitter molecule. Thus, expressed in terms of nucleotide units, the emitter molecule and the quencher are attached, incorporated and / or conjugated to the oligonucleotide at a distance of at least 12 nucleotide units in length. This distance corresponds to a distance of at least 5, 6 or 7 nm for most FRET pairs, preferably at least 9 nm for most FRET pairs. These distances are measured or expressed with respect to the FRET oligonucleotide in a linear, fully extended form or in a bound conformation.

[0130] For example, the Förster radius for FRET oligos using Atto425 / Atto488 is 5.2 nm. For FRET oligos using Atto488 / Atto550, the Förster radius is 6.3 nm. For FRET oligos using Atto550 / Atto647, the Förster radius is 6.5 nm. For FRET oligos containing Atto647 / BHQ3 (dark quencher), the Förster radius is 7.0 nm.

[0131] This can be achieved by positioning the fluorescent emitter molecule and the quencher at positions separated by at least 12 nucleotide units within the FRET oligonucleotide. For example, if the FRET oligonucleotide is 12 oligonucleotides or longer, the two molecules may be positioned such that the fluorescent emitter molecule is located at the 5'-most oligonucleotide unit of the FRET oligonucleotide and the quencher is located at the 3'-most oligonucleotide unit of the FRET oligonucleotide, or vice versa. Alternatively, the two molecules may be positioned on the FRET oligonucleotide such that they are located at least 11 oligonucleotide units apart (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 oligonucleotide units apart). For example, two molecules are separated by 11 oligonucleotide units, such as at nucleotide units 1 and 12, 1 and 13, 1 and 14, 1 and 15, 1 and 16, 1 and 17, 1 and 18, 1 and 19, 1 and 20, 2 and 13, 2 and 14, 2 and 15, 2 and 16.

[0132] In certain embodiments, at least one of the fluorescent emitter molecule and the quencher, and preferably both, are located at the terminal oligonucleotide units, for example, the fluorescent emitter molecule may be located at the 5'-most oligonucleotide unit of the FRET oligonucleotide and the quencher may be located at the 3'-most oligonucleotide unit of the FRET oligonucleotide, or vice versa.

[0133] The FRET oligonucleotide preferably has a sequence that does not form a secondary structure such as a hairpin or hairpin loop. This is because it is energetically favorable for the FRET oligonucleotide to form this secondary structure in solution. The presence of secondary structure reduces the rate at which dimers with the T oligonucleotide can be formed. The FRET oligonucleotide preferably has a sequence that does not allow self-ligation or cross-dimerization with other FRET oligonucleotides that can be used in the method of the present invention. Self-ligation or cross-dimerization with other FRET oligonucleotides that can be used in the method of the present invention produces fluorescence in the absence of hybridization to the T oligonucleotide. The FRET oligonucleotide must also not be complementary to the DNA sequence present in the sample.

[0134] The fluorescent emitter molecule and quencher are, in certain embodiments, located at the 5'-most and 3'-most oligonucleotide units of the FRET oligonucleotide. The fluorescent emitter molecule and quencher can be positioned such that the fluorescent emitter molecule is located at the 5'-most oligonucleotide unit of the FRET oligonucleotide and the quencher is located at the 3'-most oligonucleotide unit of the FRET oligonucleotide, or vice versa.

[0135] Fluorescence Emitter Molecules and Quenchers A fluorescent emitter molecule is a molecule that produces emission at a defined fluorescent wavelength upon illumination with appropriate spectral emission.

[0136] A quencher as referred to herein is a molecule to which the emission of a defined fluorescent wavelength emitted by a fluorescent emitter molecule is transferred, for example, by Förster resonance energy transfer (FRET). If a quencher is present, for example, in a suitable position, an excited fluorescent emitter molecule can return to the ground state without emitting light by transferring its energy to the quencher, while the quencher is activated to the excited state. During the emitter's excited state, energy is transferred from the emitter (or donor) molecule. FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor and acceptor, and is proportional to the 1 / R 6 is highly dependent on the distance R between the donor and the acceptor, which decays at a rate of

[0137] A fluorescent emitter molecule is activated to an excited state when excited by irradiation with an appropriate spectral emission (e.g., a particular wavelength). In the absence of a quencher, the excited molecule emits light at its emission wavelength upon returning to the ground state. When a quencher is present in an appropriate location, energy transfer to the quencher results in reduced or eliminated fluorescence emission at the emission wavelength of the emitter molecule. Depending on the nature of the quencher, energy transfer may not result in fluorescence emission (e.g., when a dark quencher or black hole quencher is used). Alternatively, a quencher may be used that causes light emission at a wavelength different from the emission wavelength, but still results in reduced or eliminated fluorescence emission at the emission wavelength of the emitter molecule.

[0138] For energy transfer (e.g., by FRET) between two molecules to occur, the spectra of the donor and acceptor must overlap appropriately, and the emission spectrum of the emitter molecule must overlap with the absorption spectrum of the quencher entity. Therefore, only certain pairs of fluorescent emitter molecules and quenchers can be used. An optimal FRET pair can be designed at any wavelength, as long as the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor. The optimal excitation wavelength of the illumination source should be based on the fluorescence emission of the FRET oligonucleotide during hybridization.

[0139] Suitable pairs of fluorescent emitter molecules and quenchers can be selected.Examples of suitable pairs include Atto425 / Atto488 (e.g., excite with UV irradiation source and detect blinking at Atto425 emission wavelength), Atto488 / Atto550 (e.g., excite with blue irradiation source and detect blinking at Atto488 emission wavelength), Ato550 / Atto647 (e.g., excite with green irradiation source and detect blinking at Atto550 emission wavelength), Atto647 / BHQ3 (dark quencher) (excite with red irradiation source and detect blinking at Atto647 emission wavelength), Cy5 fluorophore and BHQ-3 dark quencher (e.g., excite with 639nm laser and detect blinking at Cy5 emission wavelength).

[0140] BHQ-1, BHQ-2 and BHQ-3 are well known and widely used black hole quenchers in the art. See, for example, https: / / www.sigmaaldrich.com / technical-documents / articles / biology / black-hole-quenchers.html. Other dark quenchers include Dabsyl (dimethylaminoazobenzenesulfonic acid), which absorbs in the green spectrum and is often used with fluorescein, Qxl quenchers (spanning the entire visible spectrum), Iowa black FQ (absorbing in the green to yellow portion of the spectrum), Iowa black RQ (blocks the orange to red portion of the spectrum), and IRDye QC-1 (quenches dyes in the visible to near infrared range (500-900 nm)).

[0141] Other examples of FRET probe pairs are described in Yoshiyuki Arai and Takeharu Nagai (2013) Extensive use of FRET in biological imaging, Microscopy 0(0):1-10(2013), and for example, the following emitter molecules and quenchers are suitable examples. [Table 3-1] [Table 3-2]

[0142] In this specification, the emission wavelength of the emitter molecule is referred to. It is well known that the emission from an emitter molecule can be represented as a spectrum in which the emission occurs at multiple wavelengths around a peak wavelength or emission maximum. The exact shape of this spectrum and the emission maximum can be easily determined for each emitter molecule. When referring to the emission wavelength of an emitter molecule, this generally means one or more wavelengths at or near the emission maximum of the emitter molecule (e.g., the emission maximum of the emitter molecule, or a window within 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm above or below the emission maximum of a given emitter molecule).

[0143] In practice, for a given emitter molecule, its fluorescence is observed in a channel that is a predefined window of wavelengths selected based on the emission spectrum of the emitter molecule. The channel can be, for example, within 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm above or below the emission maximum of a given emitter molecule. Thus, for example, when referring to the observation or detection of a kinetic fluorescence profile at the emission wavelength of an emitter, or its presence or absence, or its reduction, this can refer to the fluorescence at the emission maximum of the emitter molecule, or the fluorescence within a predefined window surrounding the emission maximum of the emitter molecule.

[0144] Those skilled in the art can select one or more suitable channels for observing the emission of a given emitter molecule(s). The size of the channel can be easily determined and adapted to the specific experimental parameters. Generally, when a given setup is used in which a single channel is used to observe the fluorescence emission, this will be a larger window than the window for an experimental setup in which multiple channels are used. Those skilled in the art also understand to use suitable parameters, for example, to avoid the overlap of absorption and emission spectra. Depending on the nature of the one or more emitter molecules used, any suitable channel can be selected using conventional methods to observe the required fluorescence kinetics profile. For example, when a single emitter molecule is used, a single channel can be used, in which case the window can be relatively wide (e.g., within 30 nm, within 40 nm, within 50, within 50 nm, within 70 nm, within 80 nm, within 90 nm, within 100 nm). When fluorescence kinetic profiles from multiple emitter molecules are used, each window will generally be narrower, e.g., within 1 nm, within 2 nm, within 3 nm, within 4 nm, within 5 nm, within 10 nm, within 15 nm, within 20 nm, within 25 nm, within 30 nm, e.g., to avoid overlap between the various spectra. Fluorescence kinetic profiles can be observed or detected at any single wavelength or group of wavelengths within the excitation spectrum of the emitter molecules.

[0145] irradiation The method includes a step of irradiating the fluorescence emitter molecule with an appropriate spectral emission, thereby causing the fluorescence emitter molecule to generate an emission at its predefined fluorescence wavelength. This is sometimes called "excitation" and suitable wavelengths can be selected for this purpose based on the known properties of the fluorescence emitter molecules used or a combination thereof. If multiple fluorescence emitter molecules are used, a single light source can be used to excite them. Alternatively, a separate light source can be used for each fluorescence emitter molecule used, or a combination thereof. Fluorescence emitter molecules have well-characterized excitation or absorption spectra, and it is routine to select a suitable wavelength or combinations thereof for excitation of all the fluorescence emitter molecules.

[0146] Transient hybridization and fluorescence kinetic profiles As explained above, the T oligonucleotide and the FRET oligonucleotide pair are not completely complementary, but are transiently hybridized. That is, dissociation and reassociation occur between the two components of the pair. This dissociation and reassociation between the pair generates a fluorescence kinetic profile. As a result of the sequence difference between the T oligonucleotides in a set, the fluorescence kinetic profile is unique to that pair within that set.

[0147] There are several components to a fluorescence kinetic profile. Only in the hybridized state do the fluorescence emitter molecule and the quencher separate sufficiently to prevent quenching of the emission at the emission wavelength of the FRET oligonucleotide emitter molecule, so that dissociation of the pair causes the fluorescence signal at the emission wavelength of the FRET oligonucleotide emitter molecule to be "off" and reassociation between the pair causes the signal at the emission wavelength of the FRET oligonucleotide emitter molecule to be "on". A fluorescence kinetic profile can be characterized in several different ways. Any number of numerical metrics or features can be extracted from the fluorescence kinetic profile, including spatiotemporal information. Non-limiting examples of these metrics include: (i) the average time between each fluorescence emission (also called the off time), (ii) the average duration of the fluorescence emission (also called the on time), and (iii) the incidence of the fluorescence emission. At least one, at least two, or at least three such metrics can be extracted from and used to describe the fluorescence kinetic profile. Metrics other than these exemplary metrics may also be used.

[0148] Furthermore, the off and on fluorescence emission, conceptualized as "blinking", can be used to define a numerical metric that describes the hybridization affinity between each T oligonucleotide and the corresponding FRET oligonucleotide. From the blinking kinetics, any number of metrics can be derived, including at least (i) Kon (association rate constant, expressed in M-1.s-1) and (ii) Koff (dissociation rate constant, expressed in s-1). Since each T oligonucleotide is engineered to exhibit a unique hybridization kinetic profile, and thus a unique blinking kinetic profile, any individual metric or combination of metrics can be used to distinguish the identity of the T oligonucleotide under observation. These affinity metrics can also be a way to describe the fluorescence kinetic profile.

[0149] The number of occurrences of fluorescence emission at the emission wavelength of the FRET oligonucleotide emitter molecule in a given time frame means the number of times that the fluorescence emission switches on at the emission wavelength of the FRET oligonucleotide emitter molecule in a given time frame. The transient nature of the hybridization between the two components of the pair means that the fluorescence emission at the emission wavelength of the FRET oligonucleotide emitter molecule appears and disappears over time, and the emission appears at the emission wavelength of the FRET oligonucleotide emitter molecule when the pair is in the hybridized form. The number of occurrences of fluorescence emission, or the number of peaks of fluorescence intensity, can be expressed as the number per unit of time, for example per second (S). Assuming a standard concentration of FRET oligonucleotide, a higher blinking rate usually indicates low affinity hybridization between the FRET oligonucleotide and the T oligonucleotide, such as when the T oligonucleotide contains a large number of mismatches. Conversely, assuming a standard concentration of FRET oligonucleotide, a low blinking number indicates high affinity hybridization.

[0150] The profile can be described in different ways by different metrics, including non-limiting examples: (i) the average time between each fluorescence emission (also called off time), (ii) the average duration of the fluorescence emission (also called on time), and (iii) the incidence of the fluorescence emission. The on time is the duration of the fluorescence emission and can also be expressed as the bound or bright time. This parameter is specific to the nature of the hybridization between the FRET oligonucleotide and the T oligonucleotide. This is because the ability of the two components of the pair to remain hybridized after forming a pair depends on the sequence of the overlap region. In contrast, the off time (also called the unbound / dark time), which is the duration during which no fluorescence emission is present, depends on the sequence of the overlap region, but also on the concentration of the FRET oligonucleotide added to the immobilized T oligonucleotide. This is because the possibility of the two members of the pair hybridizing depends not only on the strength of their interaction but also on the probability that the two members will encounter each other.

[0151] As described above, the kinetics of the FRET oligonucleotide and the T oligonucleotide are expressed by their dissociation (unbinding) and association (binding) rate constants, K off and K on In this case, K on is affected by the concentration of the FRET oligo and its sequence (e.g., the presence of a hairpin reduces the K on ), whereas K off is affected by the stability of hybridization. Thus, for example, the longer the overlap region, the greater the K off becomes smaller (unbinding occurs more slowly). Introduction of a mismatch destabilizes the hybridization and thus reduces the K off increases (dissociation occurs rapidly).

[0152] Certain metrics that can be used to describe the fluorescence kinetic profile, such as the number of occurrences (peaks of fluorescence intensity) within a specified time frame, on-time and off-time for each oligonucleotide pair at a given concentration of T oligonucleotide and FRET oligonucleotide, can be experimentally measured and / or calculated by immobilizing the T oligonucleotide, contacting the immobilized T oligonucleotide with the FRET oligonucleotide, and observing the fluorescence kinetic profile of each immobilized T oligonucleotide. Information regarding at least one metric for each given pair forms part of the process of assigning an identity to one or more pixels in the observed sample. This assignment can be based on at least one metric of the observed and / or calculated fluorescence kinetic profile of the sample at that pixel.

[0153] It is possible to perform this deconvolution based on one metric. In certain embodiments, at least one, at least two, at least three, at least four metrics of the fluorescence kinetic profile are used. In general, the method can be more efficient by using multiple metrics, but a metric can be used alone as long as the metric used is distinguishable (e.g., in that identity can be assigned to T oligonucleotide based on that metric). In certain embodiments, it may be necessary to rely on multiple metrics, and the combined metric can be distinguishable from the combination of metrics for another given fluorescence kinetic profile.

[0154] The specific components of the fluorescence kinetic profile can also be investigated, for example, during the oligonucleotide set design process, by determining the fluorogenicity profile in solution when increasing concentrations of T oligonucleotides are incubated with the FRET oligonucleotide, for example, as shown in Example 2. In solution, hybridization of the FRET oligonucleotide to each T oligonucleotide produces a unique fluorogenicity profile, with more disruptive mismatches resulting in an apparent reduction in fluorogenicity.

[0155] In fact, when carrying out the method of the present invention, the T oligonucleotide is immobilized on the sample by the binder to which the T oligonucleotide is conjugated. The immobilization of the binder-T oligonucleotide conjugate means that the fluorescence kinetic profile at any one or more pixels can be observed. Thus, by immobilizing the T oligonucleotide and adding a FRET oligonucleotide to the immobilized T oligonucleotide, it is possible to observe the fluorescence kinetic profile at the emission wavelength of the FRET oligonucleotide emitter molecule of a particular FRET oligonucleotide T oligonucleotide pair before using such a pair in the method of the present invention. In this way, the fluorescence kinetic profile of each pair can be observed and it can be confirmed that the set of T oligonucleotides generates a suitable fluorescence kinetic profile that is useful in the method of the present invention, and in particular, that within the set, there is a fluorescence kinetic profile that is unique to the pair. Furthermore, the nature of the profile of each pair can be determined (including, for example, observing or calculating one or more metrics of the fluorescence kinetic profile).

[0156] observation Compatible with standard microscope bases and illumination systems, this technique allows the use of standard fluorescence microscopy optical systems. Image capture over time is required to observe the fluorescence profile emanating from each pixel of the observed sample. Any type of imaging device capable of detecting single molecules is suitable for this task, but a high frame rate (>100 frames per second) complementary metal oxide semiconductor (CMOS) camera can be used, as a high frame rate increases the accuracy of profiling the fluorescence kinetic profile.

[0157] For each sample, images can be observed and / or recorded over time, for example with a total capture time of up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 seconds, e.g., 10-120, 20-110, 30-100, 40-80, 50-70 seconds, preferably 0-2, 2-5, 2-10 seconds. Time traces of fluorescence intensity can be generated to show the kinetic profile of a particular region of interest within the image stack.

[0158] Image capture is generally performed in a channel. As used herein, a channel refers to a predefined window of wavelengths that includes the emission wavelength(s) of a FRET oligonucleotide emitter molecule and is selected based on the emission spectrum of the emitter molecule. A channel can be, for example, within 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm above or below the emission maximum of a given emitter molecule. As also mentioned above, the size of a channel depends on the emitter molecule or combination of emitter molecules used. For example, when referring to the observation or detection of a kinetic fluorescence profile at the emission wavelength of an emitter, or its presence or absence, or its reduction, this can refer to the fluorescence at the emission maximum of the emitter molecule, or the fluorescence within a predefined window surrounding the emission maximum of the emitter molecule. For example, if the emitter molecule used is Atto550, the emission channel may correspond to the spectral bandwidth of red emission, if the emitter molecule used is Atto647, the emission channel may correspond to the spectral bandwidth of near-infrared emission, if the emitter molecule used is Atto488, the emission channel may correspond to the spectral bandwidth of green emission.

[0159] Additionally, the fluorescence kinetic profile may be recorded, e.g., for future analysis. This may be done using any suitable technique known in the art. Additionally, the fluorescence kinetic profile may be analyzed. For example, at least one metric, as described elsewhere, may be derived or calculated from the observed and / or recorded fluorescence kinetic profile.

[0160] Image Processing A time-lapse image sequence is a set of consecutive, ordered captured images. For example, in a time-lapse image sequence, a fluorescence kinetic profile is observed for each focal point. This can then be analyzed to derive or calculate at least one metric, for example, as described elsewhere. Any metric, such as any distinguishable metric of a fluorescence kinetic profile (including, but not limited to, one or more of the average occurrences (peaks of fluorescence intensity), average on-time, and average off-time within a specified time frame can be measured or determined), can be used to assign identity to one or more pixels in the observed sample based on the metric calculated from the fluorescence kinetic profile of the sample at that pixel.

[0161] The focus is the area in which blinking emanating from a single molecule is observed. Typically, its diameter is about 3-5 pixels, but this can vary depending on the magnification and optics used.

[0162] For pixels or foci where no binder-T oligonucleotide conjugate is immobilized (i.e., background pixels), stochastic fluorescence may be observed, but the fluorescence kinetic profile of this background "noise" is highly distinguishable from the fluorescence kinetic profile of the target FRET oligonucleotide T oligonucleotide, as shown, for example, in Example 2. Examples of these representative base profiles for three different pairs are shown in Figures 4A (lower profile) and 5A (lower left profile). Figure 13 also contains examples of representative profiles.

[0163] Since each different FRET oligonucleotide T oligonucleotide pair generates a fluorescence kinetic profile that is unique to that pair within the set, the fluorescence kinetic profile (e.g., defined in terms of its at least one metric) at each pixel can be assigned an identity (e.g., to the particular T oligonucleotide that generated it). In this way, an identity is assigned to a pixel based on the fluorescence kinetic profile (e.g., defined in terms of its at least one metric) of the sample at the pixel. This is shown, for example, in FIG. 5. A cell sample contacted with antibodies that recognize different molecular epitopes, each antibody species conjugated to a different T oligo, was contacted with a FRET oligo corresponding to the conjugated T oligo. By image capture over time, the fluorescence of each pixel can be measured and a profile can be observed or created, as shown in 5B. Since each FRET oligo / T oligo pair has a unique kinetic binding profile, this profile (e.g., defined in terms of its at least one metric) can be used to associate the fluorescence profile of the pixel with the identity of its T oligo and, therefore, the cellular target. (C) Each pixel is assigned to a specific T oligo identity, or background, and (D) from this assignment, separate channel images for each imaging modality can be generated, despite the experiment using a single laser excitation wavelength. This means that it is possible to assign an identity to one or more pixels in the observed sample based on the fluorescence kinetics profile of the sample at that pixel (e.g., defined in terms of at least one metric thereof).

[0164] The fluorescence kinetic profiles (e.g., defined in terms of at least one metric thereof) of various pairs of FRET oligonucleotides T oligonucleotides can be determined experimentally, for example as described elsewhere herein, such that when any fluorescence kinetic profile is observed in a sample, it is possible to compare the observed fluorescence kinetic profile (e.g., defined in terms of at least one metric thereof) with known or predefined fluorescence kinetic profiles (e.g., defined in terms of at least one metric thereof) of the various pairs to determine which pairs are present at each position (and thus which T oligonucleotides are present in the pair, and thus which binding agents are bound at that position).

[0165] This comparison and assignment can be performed by any suitable means. For example, at least one metric of the fluorescence kinetic profile is calculated from the fluorescence kinetic profile. This metric(s) can be compared with the known or predefined metric(s) of the fluorescence kinetic profile for each pair of FRET oligonucleotides T oligonucleotides used. This comparison can assign identity to one or more pixels in the observed sample based on the metric calculated from the fluorescence kinetic profile of the sample at that pixel.

[0166] The pixel identities can then be used to generate one or more images of the sample, e.g., showing the location of one or more binding agents and / or target molecules within the sample. The pixels assigned to each T oligonucleotide identity can be separated into individual grayscale channels. The individual grayscale channel images can be combined into a pseudocolor microscope image in which each T oligonucleotide channel is shown in a different color.

[0167] Multiplexing by Sets In certain embodiments, step a comprises contacting the sample with a set of binder-T oligonucleotide conjugates, the set comprising a first set of binder-T oligonucleotide conjugates and one or more subsequent sets of binder-T oligonucleotide conjugates, and step b comprises contacting the sample and any binders bound as a result of step a with a first FRET oligonucleotide and one or more subsequent FRET oligonucleotides, the first FRET oligonucleotide being capable of hybridizing to a plurality of T oligonucleotides in the first set to form a plurality of pairs, and each of the subsequent FRET oligonucleotides being capable of hybridizing to a plurality of T oligonucleotides in its corresponding set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is (i) unique to that pair within each set, and (ii) unique to that pair within the set.

[0168] In such an embodiment, a set group is used, comprising a first set of binder-T oligonucleotide conjugates and one or more subsequent sets of binder-T oligonucleotide conjugates.Thus, a FRET oligonucleotide is used for each set of binder-T oligonucleotide conjugates.As explained above, the use of multiple FRET oligonucleotides means that a wider range of fluorescence kinetic profiles can be generated.

[0169] The set may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or at least ... , 23, 24, 25, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, for example, 1-25, 2-24, 3-23, 4-22, 5-21, 6-20, 7-19, 8-18, 9-17, 10-16, 11-15, 12-14 sets.

[0170] When a set of binder-T oligonucleotide conjugates is used, each corresponding FRET oligonucleotide may contain an emitter molecule with overlapping emission spectrum (so that the emitted fluorescence can be detected in a single channel, e.g., they may contain emitter molecules that emit the same wavelength of fluorescence or may contain the same emitter molecule). This may be advantageous, since it allows the observation step to be performed simultaneously for all oligonucleotide pairs. As an example, when a set of binder-T oligonucleotide conjugates is used, each different FRET oligonucleotide may contain an emitter molecule that is Atto550. In an alternative embodiment, each different FRET oligonucleotide may contain an emitter molecule that is Atto647. In a further alternative embodiment, each different FRET oligonucleotide may contain an emitter molecule that is Atto488.

[0171] Alternatively, when a set of binder-T oligonucleotide conjugates is used, the corresponding FRET oligonucleotide may contain emitter molecules that emit fluorescence that can be detected in multiple channels, for example, emitter molecules that emit fluorescence of different wavelengths, or may contain different emitter molecules. For example, when a set of binder-T oligonucleotide conjugates is used, One or more of the FRET oligonucleotides can include an emitter molecule that is Atto550 and one or more of the FRET oligonucleotides can include an emitter molecule that is Atto647; or One or more of the FRET oligonucleotides can include an emitter molecule that is Atto550 and one or more of the FRET oligonucleotides can include an emitter molecule that is Atto488; or Alternatively, one or more of the FRET oligonucleotides can include an emitter molecule that is Atto647 and one or more of the FRET oligonucleotides can include an emitter molecule that is Atto488; One or more of the FRET oligonucleotides can include an emitter molecule that is Atto550, one or more of the FRET oligonucleotides can include an emitter molecule that is Atto488, and one or more of the FRET oligonucleotides can include an emitter molecule that is Atto647.

[0172] Regardless of the exact method of generating multiple fluorescence kinetic profiles, it will be appreciated that creating sets of T oligonucleotides and corresponding FRET oligonucleotides increases the number of different fluorescence kinetic profiles that can be generated. A higher degree of multiplexing is possible by using sets of T oligonucleotides and corresponding FRET oligonucleotides than by using a single set of T oligonucleotides and a corresponding single FRET oligonucleotide.

[0173] Even when a set of T oligonucleotides is used, the sequence of each T oligonucleotide present is unique to the binding agent to which it is conjugated.Furthermore, the dissociation and reassociation between each different pair (i) generates a fluorescence kinetic profile that is unique to that pair within that set, and (ii) that is unique to that pair within that set.Furthermore, in some embodiments, each T oligonucleotide hybridizes only to its corresponding FRET oligonucleotide, and does not hybridize to the FRET oligonucleotide(s) of other sets within that set.Cross-hybridization should be avoided so that the correlation between the fluorescence kinetic profile, the T oligonucleotide, and the binding agent is maintained.

[0174] Multiplexing with multiple sets As described above, the method may include an embodiment in which step a includes contacting the sample with a plurality of sets of binder-T oligonucleotide conjugates, each set including a first set of binder-T oligonucleotide conjugates and one or more subsequent sets of binder-T oligonucleotide conjugates, and the emission spectrum of the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides may be detected in a channel specific for that set, and the step of observing the kinetic fluorescence profile of the sample includes observing the kinetic fluorescence profile in a channel for each set.

[0175] This is useful for achieving an additional level of multiplexing compared to a single set or set. In this way, for example, FRET oligonucleotides containing fluorophores with different emission spectra are used to generate fluorescence kinetic profiles that can be detected in different channels, thereby increasing the number of different channels in which fluorescence kinetic signals are observed.

[0176] In such cases, each of the fluorescence kinetic profiles generated may be different, but the use of multiple channels (i.e., emitter molecules that emit fluorescence at different wavelengths, or they may contain different emitter molecules) means that a given fluorescence kinetic profile can be replicated between sets. In other words, two or more pairs that generate similar or even identical fluorescence kinetic profiles can be used if they generate fluorescence kinetic profiles as fluorescence emissions observed in different channels.

[0177] In this type of multiplexing, each T oligonucleotide still has a different sequence that is unique to the binding agent that it is conjugated to, and each different pair in the set generates a fluorescence kinetic profile that is unique to that pair in the set.Since multiple sets are used and the FRET oligonucleotides of different sets generate fluorescence kinetic profiles as fluorescence emission that can be detected in different channels, this means that if a pair in one set (or a set) has a similar or identical fluorescence kinetic profile to a pair in a different set (or a set), both pairs can be used as long as the emitter molecules on the FRET oligonucleotides produce distinguishable emissions.

[0178] To cause the excitation of the emitter molecule of each FRET oligonucleotide, the sample needs to be irradiated with one or more wavelengths. Depending on the specific emitter molecule or combinations used, it may be necessary to use different irradiation sources, such as different wavelengths, to excite the emitter molecules, or a single light source may be sufficient to excite all of the emitter molecules. As an example, UV irradiation can be used to cause the excitation of each of Atto 594, Atto 700, and Atto 465.

[0179] As an example, the FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto550, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto647, or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto550, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto488; or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto647, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto488; or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto550, the FRET oligonucleotide for a second set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto488, and the FRET oligonucleotide for a third set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto647.

[0180] Alternative multiplexing with multiple sets In another embodiment of the method, step a comprises contacting sample with a plurality of sets of binder-T oligonucleotide conjugates, and the emission spectrum of the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in the channel specific to that set.In such an embodiment, a plurality of sets of binder-T oligonucleotide conjugates are used, and each set of binder-T oligonucleotide conjugates has a corresponding FRET oligonucleotide that produces a fluorescence emission detectable in a single channel, for example, each FRET oligonucleotide has an emitter molecule that emits fluorescence at a wavelength different from each other FRET oligonucleotide, for example, each FRET oligonucleotide has a different emitter molecule.

[0181] As an example, the FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto550, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto647, or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto550, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto488, or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto647, and the FRET oligonucleotide for the second set of binder-T oligonucleotide conjugates can include an emitter molecule that is Atto488, or The FRET oligonucleotide for one set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto550, the FRET oligonucleotide for a second set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto488, and the FRET oligonucleotide for a third set of binder-T oligonucleotide conjugates can comprise an emitter molecule that is Atto647.

[0182] Multiplexing using mixtures of sets and groups of sets In other embodiments of the method, mixtures of sets and sets may be used. For example, Step a comprises: i. one or more sets of binder-T oligonucleotide conjugates, where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific for that set; and ii. one or more sets of binder-T oligonucleotide conjugates, where the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to that set; and contacting the The step of observing a kinetic fluorescence profile of the sample includes observing a kinetic fluorescence profile of the sample in a channel for each excitation / emission spectrum.

[0183] kit Also provided is a kit for multiplexed fluorescence microscopy, comprising a set of binder-T oligonucleotide conjugates, the set comprising multiple binders with different specificities, the sequence of the T oligonucleotide being unique to the binder to which it is conjugated, and a FRET oligonucleotide, the FRET oligonucleotide being capable of hybridizing to multiple T oligonucleotides in the set to form multiple pairs, the dissociation and reassociation between each different pair generating a fluorescence kinetic profile unique to that pair within the set. The components of this kit can be used to carry out the method of the invention. The kit may further comprise suitable buffers and solutions for carrying out the method steps, such as fixation reagents as defined herein and / or permeabilization reagents as defined herein, and / or suitable solutions and liquids for washing steps.

[0184] The kit may further comprise one or more additional sets of binder-T oligonucleotide conjugates and one or more additional FRET oligonucleotides, each of which is capable of hybridizing to a plurality of T oligonucleotides in its corresponding set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile unique to that pair within each set.

[0185] Thus, the kit may comprise one or more sets of binder-T oligonucleotide conjugates or one or more groups of sets, for example as defined elsewhere herein in the context of the present method, and a corresponding number of FRET oligonucleotides.Each FRET oligonucleotide can hybridize to multiple T oligonucleotides in its corresponding set to form multiple pairs, and the dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within each set.The emission spectrum of the FRET oligonucleotide can be specific to that set or groups of sets, and can be different between sets and groups of sets.

[0186] The present invention also provides one or more sets of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides for producing a kit for multiplexed fluorescence microscopy, comprising one or more sets of T oligonucleotides and one or more FRET oligonucleotides, where the set of T oligonucleotides and the one or more FRET oligonucleotides are as defined elsewhere herein.

[0187] A preferred set of T oligonucleotides and corresponding FRET oligonucleotides include: - A set containing (or possibly consisting of) the following: o SEQ ID NO:1 (5'-TTCCACATTACTTCT-3') and SEQ ID NO: 2 (5'-ATCCCCATTACTTCT-3') one or both of the T oligonucleotides of the sequence And o the corresponding FRET oligonucleotide of sequence SEQ ID NO: 7 (5'-AGAAGTAATGTGGAA-3'), - A set containing (or possibly consisting of) the following: o SEQ ID NO: 3 (5'-GGGTGGCGTATGATAGCTAT-3'), SEQ ID NO: 4 (5'-AGGTTAAGTATGATAGTTAT-3'); SEQ ID NO:5 (5'-GGGTTAAGCATGGTAGTTAT-3') and SEQ ID NO:6 (5'-AGGTTAAGCGTTATATTTAT-3') at least one of the T oligonucleotide(s) of the sequence (e.g., at least two, three, or all four of the listed T oligonucleotides); And o The corresponding FRET oligonucleotide of sequence SEQ ID NO: 8 (5'-ATAACTATCATACTTAACCT-3').

[0188] The above two sets can be used together as a set, for example, when two corresponding FRET oligonucleotides contain emitter molecules with overlapping emission spectra, and optionally have the same emitter molecule.

[0189] How to prepare the kit The present invention also provides a method for preparing the kit mentioned above, wherein each set of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides are provided.The method further comprises conjugating a set of T oligonucleotides with a set of binders to form a set of binder-T oligonucleotide conjugates, wherein the set of binder-T oligonucleotide conjugates comprises a plurality of binders with different specificities, and the sequence of T oligonucleotide is unique to the binder it is conjugated to within the set.The conjugation step can be carried out by any suitable means known in the art, such as those described above.

[0190] Sets of T oligonucleotides and methods for designing FRET oligonucleotides for use in the methods of the invention Generation of FRET oligonucleotides: Generally, FRET oligonucleotide is generated first, and then T oligonucleotide sequence is generated.FRET oligonucleotide generally has a nucleotide sequence that is not complementary to any sequence found in the sample that the method is applied to.This is to avoid the hybridization of FRET oligonucleotide with the nucleic acid material in sample, which causes fluorescence emission in the absence of the binding agent-T oligonucleotide conjugate that binds to target molecule in sample.

[0191] A suitable process for generating FRET oligonucleotides is as follows: An initial sequence is selected from sequences randomly generated containing combinations of simple to more complex nucleotides and pre-filtered to avoid matching any known genomic sequences. A suitable sequence provides sufficient flexibility to bring the termini in close proximity in the unbound state of the FRET oligonucleotide, for example to participate in electron transfer or exchange.

[0192] Since there should be diversity in the fluorescence kinetic profile of the pairs used in the present method, the length of the FRET oligonucleotide can be selected based on the desired length of time that it remains bound (i.e., associated with the T oligonucleotide). Increasing the length of the FRET oligonucleotide strengthens base pairing, so that it takes longer or requires more energy to dissociate. Increasing its GC content strengthens base pairing, so that it takes longer or requires more energy to dissociate.

[0193] Once a suitable sequence for the FRET oligonucleotide is generated in silico, the sequence is tested to ensure the absence of secondary structures and the possibility of self-ligation and / or cross-dimerization with other FRET oligos used in the method. This can be done by methods known in the art, such as standard software tools provided by most oligonucleotide suppliers. Examples of commercially available tools include, for example, Sigma-Aldrich's "Oligo Analyzer Tool," Thermofisher's "Multiple Primer Analyzer," and IDT's "OligoAnalyzer™ Tool."

[0194] Generation of T oligonucleotides: Once the FRET oligonucleotide sequence has been generated, a suitable T oligonucleotide can then be generated. Typically, this is done by starting with a sequence that is perfectly complementary to all or part of the FRET oligonucleotide.

[0195] Possible T oligonucleotides are generated in silico by introducing mismatches into the perfectly complementary sequence. For example, mismatches can be introduced into 20-40% of the possible base pairs. Mismatches can be introduced in different patterns, as explained above.

[0196] The possible fluorescence profile kinetics for a given pair can be inferred from the number and positions of mismatches (e.g., a pair with a series of mismatches will have a different profile than a pair with the same number of mismatches but evenly distributed across the sequence). Sequences likely to give rise to the required fluorescence kinetic profile are synthesized and tested for their blinking profile.

[0197] This is shown, for example, in FIG. 2. The fluorogenicity profile of a FRET / T oligonucleotide pair can be evaluated in solution. The FRET oligonucleotide is contacted with different T oligonucleotides in solution, and the fluorescence emission is measured at various T oligonucleotide concentrations, and optionally compared to a perfectly matched sequence. As can be seen in FIG. 2A, the perfectly matched oligonucleotide produces a different profile than the mismatched T oligonucleotide, and different mismatched pairs have different fluorogenicity profiles in solution. These unique kinetic profiles are generated when the FRET oligonucleotide is in the presence of different T oligonucleotides that contain mismatches that interfere with proper base pairing between the FRET oligonucleotide and the T oligonucleotide. Hybridization of the FRET oligonucleotide to each T oligonucleotide produces a unique fluorogenicity profile, with more disruptive mismatches resulting in an apparent reduction in fluorogenicity.

[0198] When tested in solution, only the total amount of fluorescence can be measured. Further information on the profile can be generated by adding a FRET oligonucleotide to the same T oligonucleotide, this time immobilized on a surface (mimicking what happens in the method of the present invention, where the T oligonucleotide is conjugated to a binding agent, which is then bound to the sample, effectively immobilizing the T oligonucleotide on the sample). The FRET oligo in this configuration emits intermittent fluorescence. Figure 2B shows representative results for oligonucleotide pairs tested, with the number of blinking events per second (e.g., the number of occurrences of fluorescent emission within a specified time frame of seconds) shown for each pair. As explained above, the number of occurrences of fluorescent emission within a specified time frame is one component of the fluorescence kinetics profile. The blinking kinetics is influenced by factors including the nature of the mismatches introduced into the T oligonucleotide, such as disruptive mismatches that reduce the fluorogenic profile of the FRET oligonucleotide. As shown in Figure 2B, different pairs have different numbers of occurrences of fluorescent emission within a specified time frame.

[0199] The ability to observe the fluorescence kinetic profile of a given pair in this test format means that when designing a pair, appropriate sequences can be selected to yield the range of profiles required for a given application.

[0200] Thus, the invention provides a set of T oligonucleotides and a method for designing a FRET oligonucleotide for use in the method of the invention, the method comprising: a. selecting a FRET oligonucleotide sequence at least 12 nucleotides in length; b. obtaining one or more sequences at least 8 nucleotides in length that are complementary to the FRET oligonucleotide sequence; c. generating a plurality of potential T oligonucleotide sequences, each of which differs by at least one nucleotide from the sequence complementary to the FRET oligonucleotide; d. selecting at least two T oligonucleotide sequences based on their ability to generate fluorescence kinetic profiles that differ from one another.

[0201] Step a of selecting a FRET oligonucleotide sequence of at least 12 nucleotides in length may be performed as described above and may include one or more of: (i) determining whether the potential FRET oligonucleotide has a nucleotide sequence that is not complementary to any sequence found in the sample to which the method is applied; (ii) determining whether the sequence has sufficient flexibility to bring the termini in close proximity in the unbound state of the FRET oligonucleotide, e.g., to participate in electron transfer or electron exchange; (ii) determining whether the potential FRET oligonucleotide has a nucleotide sequence that is not complementary to any sequence of another FRET oligonucleotide used in the method of the invention.

[0202] Step b of obtaining one or more sequences of at least 8 nucleotides in length that are complementary to the FRET oligonucleotide sequence is carried out simply by making complementary pairs. Because FRET oligonucleotides are generally at least 12 nucleotide units in length and the T oligonucleotides may be shorter, this step may require generating multiple possible complementary sequences of different lengths.

[0203] Step c, generating a plurality of potential T oligonucleotide sequences, each differing by at least one nucleotide from the sequence complementary to the FRET oligonucleotide, can be performed in silico or manually, similar to step b.

[0204] Step d of selecting at least two T oligonucleotide sequences based on their ability to generate fluorescence kinetic profiles that are distinct from each other can be done by inferring these properties from the sequences, e.g., the nature, number, and location of mismatches, or by synthesizing the molecules and testing them. Testing them can include contacting members of each pair in solution and observing the fluorescence emission, e.g., determining the amount of light emitted. This can be done at multiple concentrations of potential T oligonucleotides to generate a fluorogenic profile, e.g., as shown in FIG. 2A. Additionally, potential T oligonucleotides can be immobilized on a solid surface and contacted with the FRET oligonucleotide, and the fluorescence kinetic profile of each pair can be observed.

[0205] At least two T oligonucleotides are then selected to form a set, which T oligonucleotides give rise to unique fluorescence kinetic profiles within the set.

[0206] The same principles can be applied when designing FRET oligonucleotides for inclusion in a second or subsequent set or for use in a set. Additional considerations for the second or subsequent set include (i) the need for the FRET oligonucleotide(s) not to hybridize to any T oligonucleotides in the other set, and (ii) the need not to hybridize to other FRET oligonucleotides.

[0207] A preferred set of T oligonucleotides and corresponding FRET oligonucleotides include: - A set containing (or possibly consisting of) the following: o SEQ ID NO:1 (5'-TTCCACATTACTTCT-3') and SEQ ID NO: 2 (5'-ATCCCCATTACTTCT-3') one or both of the T oligonucleotides of the sequence And o the corresponding FRET oligonucleotide of sequence SEQ ID NO: 7 (5'-AGAAGTAATGTGGAA-3'), - A set containing (or possibly consisting of) the following: o SEQ ID NO: 3 (5'-GGGTGGCGTATGATAGCTAT-3'), SEQ ID NO: 4 (5'-AGGTTAAGTATGATAGTTAT-3'); SEQ ID NO:5 (5'-GGGTTAAGCATGGTAGTTAT-3') and SEQ ID NO:6 (5'-AGGTTAAGCGTTATATTTAT-3') one or more T oligonucleotides of the sequence And o The corresponding FRET oligonucleotide of sequence SEQ ID NO: 8 (5'-ATAACTATCATACTTAACCT-3').

[0208] General rules The term "comprising" encompasses "including" and "consisting." For example, a composition "comprising" X may consist of only X or may include some additional elements, e.g., X+Y.

[0209] The term "about" in reference to a number x means, for example, x±10%.

[0210] The word "substantially" does not exclude "completely". For example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definition of the invention.

[0211] Where the invention provides a process that includes multiple consecutive steps, the steps are performed in the order shown, i.e., numerical or alphabetical order. However, those skilled in the art will appreciate that the order of the steps may be altered and still achieve useful results. The invention may also provide a process that includes less than the total number of steps. [Brief description of the drawings]

[0212] [Figure 1] DNA-based stochastic fluorescence using a Förster resonance energy transfer oligonucleotide (FRET oligonucleotide)-T oligonucleotide pair. (A) When the FRET oligonucleotide is in solution, upon illumination with the excitation wavelength of the emitter, no fluorescence is observed due to efficient energy transfer to the quencher. (B) However, hybridization of the FRET oligonucleotide with an immobilized T oligonucleotide physically separates the emitter and quencher, slowing the rate of FRET and allowing emission from the emitter fluorophore to be detected at the hybridization site. [Figure 2A] Fluorogenic and blinking profiles of FRET / T oligopairs are shown. The fluorogenic profiles of FRET / T oligopairs are evaluated in solution. Unique kinetic profiles are generated when the FRET oligonucleotides are in the presence of different T oligonucleotides that contain mismatches that interfere with proper base pairing between the FRET oligonucleotide and the T oligonucleotide. Hybridization of the FRET oligonucleotide to each T oligonucleotide generates unique fluorogenic profiles, with more disruptive mismatches resulting in an apparent reduction in fluorogenicity. The blinking kinetics is influenced by factors including the nature of the mismatches introduced into the T oligonucleotide, such that disruptive mismatches that reduce the fluorogenic profile of the FRET oligonucleotide (A) increase its blinking rate (B). [Figure 2B] Fluorogenic and blinking profiles of FRET / T oligopairs are shown. When a FRET oligonucleotide is added to the same T oligonucleotide, this time immobilized on a surface or in a cell via antibody conjugation, the intermittent fluorescence of the FRET oligonucleotide can be directly observed. The blinking kinetics is influenced by factors including the nature of the mismatches introduced into the T oligonucleotide, such that a disruptive mismatch that reduces the fluorogenic profile of the FRET oligonucleotide (A) increases its blinking rate (B). [Figure 3A] Examples of different fluorescence kinetic profiles are shown. [Figure 3B] Examples of different fluorescence kinetic profiles are shown. [Figure 3C] Examples of different fluorescence kinetic profiles are shown. [Figure 3D] Examples of different fluorescence kinetic profiles are shown. [Figure 4A] Representation of the emission signal from the binding of immobilized T oligonucleotide + FRET oligonucleotide in a cell sample. Primary antibodies that recognize the structure of specific cellular components are covalently bound to different T oligonucleotides and used for immunolabeling of fixed and permeabilized cells. Transient hybridization of the FRET oligonucleotides results in intermittent fluorescence at given foci. [Figure 4B] A representation of the emission signal from the binding of immobilized T oligonucleotide + FRET oligonucleotide in a cell sample is shown. By distinguishing the kinetics of binding hybridization (and therefore fluorescence), the identity of each focus can be assigned to one of several dozens of FRET oligonucleotide / T oligonucleotide pairs or to the background. [Figure 5A] Figure 1 shows the construction of a deconvoluted channel image of a T oligonucleotide. A cell sample is labeled with antibodies that recognize different molecular epitopes. Each antibody species is conjugated to a different T oligonucleotide. A FRET oligonucleotide corresponding to the conjugated T oligonucleotide is then added to the sample. Each FRET oligonucleotide pair uses the same emitter fluorophore and fluorescence quencher. When unbound from the T oligonucleotide, the FRET oligonucleotide cannot fluoresce due to FRET-based quenching. When hybridized to the corresponding T oligonucleotide, fluorescence occurs at the hybridization site. Image capture over time allows the fluorescence of each pixel to be measured and a profile created. [Figure 5B]Construction of a deconvoluted channel image of a T oligonucleotide is shown. Each FRET oligonucleotide / T oligonucleotide pair has a unique kinetic binding profile that can be used to relate the fluorescence profile of a pixel to the identity of that T oligonucleotide and therefore to the cellular target. [Figure 5C] A deconvoluted channel image of the T oligonucleotides is shown, where each pixel is assigned to the identity of a specific T oligonucleotide or to the background. [Figure 5D] Figure 1 shows the construction of deconvoluted channel images of the T oligonucleotide. From this assignment, separate channel images for each imaging modality can be generated, despite the experiment using a single laser excitation wavelength. [Figure 6] Shown is the dose response of Cy5-BHQ-3 FRET oligonucleotide in the presence of different T oligonucleotides. Fluorescence intensity of three replicates was averaged using either the total number of raw pixels or the average number calculated across a stack of 50 x 10 ms images captured approximately 10 μm above the bottom of the well. Values ​​were normalized to the average fluorescence value obtained for the Cy5-labeled control oligo (i.e., unquenched emitter). For the FRET oligo / perfectly matched T oligo pair (0 mismatch), the average value across all sample plates was calculated. Shown here is a plot tabulated by number of mismatches with each shaded area encompassing the 95% CI of the various profiles obtained within each group. [Figure 7A] It is shown that a wide range of fluorogenic profiles are obtained for the FRET oligo in the presence of the three mismatched T oligo, suggesting that the mismatches are not equal, with some being more costly than others. For each hybridization, the position and base substitution are shown. [Figure 7B]It is shown that a wide range of fluorogenic profiles are obtained for the FRET oligo in the presence of the three mismatched T oligo, suggesting that the mismatches are not equal, with some being more costly than others. For each hybridization, the position and base substitution are shown. [Figure 8A] The introduction of four mismatches into the T oligo generally results in a higher cost and dramatically affects the fluorogenicity profile of the FRET oligo (dose-response curves where normalized fluorescence remains below 0.1). However, the FRET oligo appears to hybridize better to some selected T oligos (curves where normalized fluorescence reaches 0.1-0.2). In comparison, fast blinking pairs in which the T oligo contains four mismatches (dotted line) are among the less fluorogenic. [Figure 8B] The introduction of four mismatches into the T oligo generally results in a higher cost and dramatically affects the fluorogenicity profile of the FRET oligo (dose-response curves where normalized fluorescence remains below 0.1). However, the FRET oligo appears to hybridize better to some selected T oligos (curves where normalized fluorescence reaches 0.1-0.2). In comparison, fast blinking pairs in which the T oligo contains four mismatches (dotted line) are among the less fluorogenic. [Figure 9A] We show that the introduction of a single mismatch in a T oligo affects the solution fluorescence profile of the FRET oligo differently depending on the position of the mismatch and its identity. For each hybridization, the position and base substitution are shown. Fluorescence profiles obtained upon incubation of the FRET oligo with increasing concentrations of the fully complementary T oligo are included. Hybridization partners with the same position but different mismatches are shown. For example, comparing substitutions at position 14 shows that a T to A substitution does not substantially affect the fluorogenicity of the FRET oligo, while a T to C substitution results in a decrease in fluorogenicity. [Figure 9B]We show that the introduction of a single mismatch in a T oligo affects the solution fluorescence profile of the FRET oligo differently depending on the position of the mismatch and its identity. For each hybridization, the position and base substitution are shown. Fluorescence profiles obtained upon incubation of the FRET oligo with increasing concentrations of the fully complementary T oligo are included. Hybridization partners with the same position but different mismatches are shown. For example, comparing substitutions at position 14 shows that a T to A substitution does not substantially affect the fluorogenicity of the FRET oligo, while a T to C substitution results in a decrease in fluorogenicity. [Figure 10] Maximum intensity Z projections for complementary "match" and mismatch T oligos showing the cumulative number and distribution of blinking events over a total capture period of 40 seconds are shown. An increasing number of spots indicates an increasing number of blinking events. The mismatch T oligonucleotide (right) produces more blinking during exposure than the match T oligonucleotide (left). [Figure 11] A workflow illustrating the fluorescence kinetic profiling of FRET oligos paired with different T oligos is shown. For each experimental group, 2000 images were captured at a frame rate of 20 ms, resulting in a total capture time of 40 seconds. The fluorescence intensity time traces (boxed traces) show the kinetic profile of the specific region of interest in the image stack to which they are associated. The histogram plots on the right show the cumulative frequency of blinking events per frame over the total capture time for the two different T oligo designs. [Figure 12A]Figure 1 shows focus kinetic profiles obtained upon addition of FRET oligos to immobilized T oligos on biotin / streptavidin coated glass coverslips. For each measurement, individual foci were recorded with 1000 x 20 ms exposures. Sample fluorescence kinetic profiles are shown for a perfectly matched T oligonucleotide (A) and a mismatched T oligonucleotide (B). The average number of blinking events during the total duration of exposure is less for the matched T oligonucleotide (1.8) compared to the mismatched T oligonucleotide (4.6). Furthermore, the average on-time of a given blinking event is longer for the matched T oligonucleotide (95 ms) versus the mismatched (30 ms). These are two exemplary metrics that can be used to deconvolute the identity of the T oligonucleotides. [Figure 12B] Figure 1 shows focus kinetic profiles obtained upon addition of FRET oligos to immobilized T oligos on biotin / streptavidin coated glass coverslips. For each measurement, individual foci were recorded with 1000 x 20 ms exposures. Sample fluorescence kinetic profiles are shown for a perfectly matched T oligonucleotide (A) and a mismatched T oligonucleotide (B). The average number of blinking events during the total duration of exposure is less for the matched T oligonucleotide (1.8) compared to the mismatched T oligonucleotide (4.6). Furthermore, the average on-time of a given blinking event is longer for the matched T oligonucleotide (95 ms) versus the mismatched (30 ms). These are two exemplary metrics that can be used to deconvolute the identity of the T oligonucleotides. [Figure 13]Spectrally independent multiplexed imaging SIMI of mismatched and complementary "matched" T oligos upon addition of FRET oligos. T oligo-coated beads in the presence of 100 nM FRET oligos. Images represent maximum intensity Z projections of 1000 exposures of 10 ms. Circles indicate beads containing complementary T oligos (A) and mismatched T oligos (B) and their corresponding kinetic profiles (right panel +). Circle C indicates an equal area used to measure background. The average signal measured within the circled area is plotted against exposure time. Beads coated with mismatched T oligos exhibit less overall fluorescence than beads coated with matched T oligos. This difference is due to the shorter blinking on time in the mismatched T oligos, as this is the same sample using a single FRET oligos. [Figure 14A] Spectrally independent multiplexed imaging (SIMI) of six cellular targets on fixed cells is shown. The targets were imaged by immobilizing six different antibodies on fixed cells, each conjugated to a different T oligo derived from two different T oligo sets. Two FRET oligos, each associated with one of the sets of T oligos, were added simultaneously and diluted to 5 nM in imaging buffer. 10,000 frames with 10 ms exposure each were captured with a Nikon 60x 1.49NA TIRF objective, filtered with a Cy5 filter set, and imaged using a Prime BSI Express CMOS camera. Super-resolution images were reconstructed from the accumulated frames using Picasso and QC-STORM software. Demultiplexing of the super-resolution images was performed using metrics derived from the fluorescence kinetics profile associated with each FRET oligo / T oligo. A super-resolution image, including the spatial location of individual molecules, was reconstructed from thousands of raw images (forming a time-lapse sequence). [Figure 14B]Spectrally independent multiplexed imaging (SIMI) of six cellular targets on fixed cells is shown. The targets were imaged by immobilizing six different antibodies on fixed cells, each conjugated to a different T oligo derived from two different T oligo sets. Two FRET oligos, each associated with one of the sets of T oligos, were added simultaneously and diluted to 5 nM in imaging buffer. 10,000 frames of 10 ms exposure each were captured with a Nikon 60x 1.49NA TIRF objective, filtered with a Cy5 filter set, and imaged using a Prime BSI Express CMOS camera. Super-resolution images were reconstructed from the accumulated frames using Picasso and QC-STORM software. Demultiplexing of the super-resolution images was performed using metrics derived from the fluorescence kinetics profile associated with each FRET oligo / T oligo. Demultiplexing of the reconstructed images allows the assignment of individual cellular targets to separate channels. Six individual grayscale channel images are shown, identifying the location of the six different cellular targets on the sample. [Figure 14C]Spectral independent multiplexed imaging (SIMI) of six cellular targets on fixed cells is shown. The targets were imaged by immobilizing six different antibodies on fixed cells, each conjugated to a different T oligo derived from two different T oligo sets. Two FRET oligos, each associated with one of the sets of T oligos, were added simultaneously and diluted to 5 nM in imaging buffer. 10,000 frames with exposures of 10 ms each were captured with a Nikon 60x 1.49NA TIRF objective, filtered with a Cy5 filter set, and imaged using a Prime BSI Express CMOS camera. Super-resolution images were reconstructed from the accumulated frames using Picasso and QC-STORM software. Demultiplexing of the super-resolution images was performed using metrics derived from the fluorescence kinetics profile associated with each FRET oligo / T oligo. Pixels are assigned to FRET oligo / T oligo pairs (and thus cellular targets) using pixel-wise association metrics calculated over a defined number of frames. Some of these metrics can be calculated from the time trace (fluorescence kinetic profile), as shown here for three example pixels. [Figure 14D]Spectrally independent multiplexed imaging (SIMI) of six cellular targets on fixed cells is shown. The targets were imaged by immobilizing six different antibodies on fixed cells, each conjugated to a different T oligo derived from two different T oligo sets. Two FRET oligos, each associated with one of the sets of T oligos, were added simultaneously and diluted to 5 nM in imaging buffer. 10,000 frames of 10 ms exposure each were captured with a Nikon 60x 1.49NA TIRF objective, filtered with a Cy5 filter set, and imaged using a Prime BSI Express CMOS camera. Super-resolution images were reconstructed from the accumulated frames using Picasso and QC-STORM software. Demultiplexing of the super-resolution images was performed using metrics derived from the fluorescence kinetics profile associated with each FRET oligo / T oligo. Features containing spatiotemporal information extracted from each pixel can be plotted to classify the pixels and assign targets (demultiplexing). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0213] Example 1 The T oligos are covalently attached to different antibodies that recognize different epitopes in the cells. The cells are fixed and permeabilized using standard cell biology protocols. The primary antibody-T oligo conjugates are contacted with the fixed and permeabilized sample and allowed to bind to their respective epitope sites. The cells are washed to remove unbound antibody-T oligo conjugates. The FRET oligos are then incubated with the sample under illumination at the excitation wavelength of the emitter fluorophore. When the FRET oligos are not hybridized to their respective T oligos, the quencher suppresses the fluorescence emission from the emitter fluorophore. Upon hybridization, this quenching is released, allowing detection of fluorescence at the hybridization site.

[0214] A high-speed CMOS sensor captures images of the sample within seconds to minutes. The fluorescence at each focal spot is monitored over time, generating a kinetic fluorescence profile. The fluorescence profile (or its metrics) at a particular focal spot is used to determine the fluorescence kinetic profile for that focal spot.

[0215] The identity of each pixel is assigned to one of the FRET oligo / T oligo pairs or to the background.

[0216] The pixels for each FRET oligo / T oligo pair are separated into individual channels, representing dozens of different imaging modalities captured under the same illumination wavelength of light.

[0217] Example 2 Example 2 - Generation of FRET and transactivating oligonucleotide pairs with unique focal kinetic profiles for use in Spectrally Independent Multiplexed Imaging (SIMI) Introduction The aim is to develop a platform for multiplex imaging by super-resolution microscopy using probes called FRET oligonucleotides or FRET oligos. These FRET oligos are designed to allow for transient binding to designated DNA targets and consequent switching (i.e., blinking) between visible and invisible states. The design utilizes quenching by FRET (Förster Resonance Energy Transfer), where the ends of the FRET oligos are conjugated with a quencher, such as a non-fluorescent quencher-acceptor, and an emitter (e.g., a fluorophore donor). The nucleotide sequence of the probe provides high structural flexibility, allowing the emitter and quencher to be in close proximity, resulting in strong quenching and suppression of background fluorescence. When the FRET oligo binds to the transactivating oligonucleotide or T oligo, a conformational change occurs that prevents energy transfer from the donor to the acceptor, and the donor fluorescence is detected.

[0218] Spectrally independent multiplexed imaging (SIMI) is in principle capable of imaging an unlimited number of intracellular targets at the nanometer length scale. SIMI using FRET oligo / T oligo pairs aims at kinetic profiling of the blinking rates associated with various oligonucleotide sequence designs. This may enable the identification of multiple target markers in a single super-resolution image capture, leading to a significant improvement in the phenomics profiling capabilities of SIMI based on FRET oligo / T oligos. To realize this goal, the kinetic profiles of different FRET oligo / T oligo pairs need to be sufficiently different from each other to be accurately identified in the same field of view.

[0219] Materials and Methods Oligo An exemplary FRET oligo is a 15 nucleotide long sequence (5'AGAAGTAATGTGGAA (SEQ ID NO: 7)) from Chung et al. BioRxiv 2020 (Chung, K. KH, Zhang, Z., Kidd, P., Zhang, Y., Williams, ND., Rollins, B., Yang, Y., Lin, C., Baddeley, D. and Bewersdorf, B. (2020). Fluorogenic probe for fast 3D whole-cell DNA-PAINT. BioRxiv) conjugated with a Cy5 fluorophore and a BHQ-3 dark quencher at the 5' and 3' ends, respectively. We used a Python script to generate T oligos by introducing mismatches at random positions. All oligos (i.e., FRET oligos and T oligos) were purchased from IDT and resuspended in 100 μM TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Aliquots were prepared to limit the number of freeze-thaw cycles and stored at -20°C.

[0220] Fluorogenic profiling T oligos were prepared at 100 μM, 10 μM, or 1 μM in TE buffer and diluted to 5, 50, 500, or 5000 nM in imaging buffer (1× PBS with 500 mM NaCl). FRET oligos were used at a final concentration of 50 nM in a total volume of 50 μL.

[0221] Buffer alone, Cy5 or BHQ-3 labeled oligonucleotide alone, or FRET oligonucleotide alone were used as controls. Additionally, samples containing FRET oligonucleotides incubated with increasing concentrations of the perfectly complementary T oligonucleotide as defined above were used as additional controls to define the intrinsic fluorogenicity profile of the FRET oligonucleotide.

[0222] Fluorescence emission from the probes was measured using an Olympus BX50 inverted microscope. Samples were illuminated using a mercury arc lamp filtered through a 530-550 nm bandpass filter. Fluorescence emission from the samples was passed through a 590 nm longpass filter and imaged onto an sCMOS camera chip. Fifty images of 10 ms exposure were captured using a 40x 0.6NA objective for each well (second integration time was 500 ms) and pixel intensities of all stack images were averaged or summed using Image J. Each sample was loaded in triplicate into a 96-well plate.

[0223] Background fluorescence from imaging wells containing only buffer or BHQ-3-labeled oligos (values ​​were comparable) was subtracted from the fluorescence intensity values ​​obtained for each sample. Reported values ​​were calculated as the average of three replicates and normalized to the fluorescence intensity value obtained with the Cy5-labeled oligos (i.e., unquenched emitters).

[0224] SIMI T oligo blinking profiling For each T oligo, 1 μM of biotinylated T oligo was added to a BSA-biotin-streptavidin coated glass coverslip mounted on an 18-well Ibidi sticky slide and incubated for 1 h at 37 °C or overnight at 4 °C. The washed coverslip was then immersed in 50 μL of imaging buffer (TE buffer with 500 mM NaCl) and placed under the microscope. 10 nM of Cy5 / BHQ-3 FRET oligo Fret probe was then added to the sample. After excitation with a 639 nm laser operating at full power (150 mW), the fluorescence was collected using a Nikon 60x 1.49NA TIRF objective, filtered with a Cy5 filter set, and imaged using a Prime BSI Express CMOS camera. Image analysis and post-processing of the captured images were performed with ImageJ, and single particle detection was performed using the open source plugin trackmate.

[0225] T oligo focal kinetic profiling 5 μL of streptavidin-coated polystyrene beads (Φ=3 μm) were resuspended in 50 μL of binding / washing buffer (20 mM Tris, pH 7.5; 1 M NaCl, 1 mM EDTA). The washed beads were then mixed with 1 μM biotinylated T oligo and incubated overnight at 4°C.

[0226] 1 μL of the mixture of mismatched and perfectly complementary T oligo-conjugated beads was immobilized on BSA / biotin coated glass coverslips of an 18-well Ibidi sticky slide and imaged in the presence of 100 nM FRET oligo using the same microscope setup as above.

[0227] A stack of 1000 10 ms exposures was used to generate blinking kinetics profiles of fast and matched T oligos.

[0228] result We designed FRET oligos using the oligonucleotide sequences published by Chung et al. BiorXiv 2019 by labeling the 5' and 3' ends with Cy5 and BHQ-3, respectively, which are dye / quencher pairs commonly used in FRET experiments.

[0229] We measured the fluorescence in solution of Cy5 / BHQ-3 FRET oligos in the presence of increasing concentrations of degraded T oligos. To generate these, we designed a Python script to modify 1-6 nucleotides at random positions in a perfectly complementary T oligo. Fifteen T oligos in each category of 1-6 mismatches were tested for fluorescence emission upon binding to the FRET oligo.

[0230] A general correlation was observed between the number of mismatches introduced into the hybridization partners and the dose-response curves obtained for each probe / partner pair (Figure 6), such that the more mismatches introduced, the lower the fluorogenicity and saturation fluorescence level (SFL) values ​​of the probe. Furthermore, with some notable exceptions, T oligos with two or more mismatches showed 3-40 times higher activation concentrations 50 (AC50) than fully complementary T oligos. This suggests that higher concentrations of partially complementary hybridization partners are required to activate FRET oligos. In general, the introduction of five or more mismatches significantly reduced the fluorogenicity of the FRET oligo probes, which could disappear completely in some cases (Figure 6). The widest range of fluorogenicity profiles was obtained with the introduction of three mismatches (Figure 7), while some T oligos with four mismatches appeared to perform better than the majority (Figure 8).

[0231] To further investigate whether specific mismatches could explain the observed differences in fluorogenicity profiles, especially for T oligos with 4 or less mismatches, the position and identity of the substitutions were analyzed. Analysis of T oligos with single mismatches and FRET oligo probes showed that certain positions affected dose response and SFL more than other positions (Figure 9). In general, the first and centrally located residues in the sequence were important for FRET oligos to bind to T oligos, while downstream positions appeared to be less important. Comparison of mismatches at similar positions but with different substitutions suggested that some residues may be more detrimental to FRET oligo / T oligo hybridization and fluorescence emission than others.

[0232] In a next step, we selected a set of T oligos with unique fluorogenicity profiles to test the hypothesis that a correlation exists between the fluorogenicity of a FRET oligo / T oligo pair and its blinking properties.

[0233] To establish a robust measurement protocol for recording blinking events, two T oligo designs were first studied: a fully complementary T oligo, denoted as "match", and a T oligo with four nucleotide mismatches, denoted as "mismatch". Image stacks consisting of 2000 x 20 ms exposures were captured in three different areas of each well containing T oligos immobilized on the surface. Analysis of the maximum intensity z-projections of the 2000 x 20 ms camera exposures for the two T oligos revealed a qualitative change in the density of events, with a significantly increased number of events in the four-mismatch T oligo compared to the fully complementary T oligo (Figure 10). By selecting regions of interest and measuring the fluorescence intensity across the image stack (Figure 11), individual time traces of the regions of interest where blinking occurs were visualized (green and purple line plots). Analysis of the cumulative distribution of the total number of events detected per frame demonstrated that the distribution shifts to the right as the blinking frequency per frame increases, allowing the identification of distinct kinetic profiles for the two T oligos.

[0234] Using this experimental platform and analytical workflow, we detected blinking events generated by Cy5-BHQ3 / Fret probes conjugated to a set of T oligos with different sequence designs and fluorogenic profiles. We observed that the changes in kinetic blinking profiles between different T oligos corresponded to the inverse fluorogenic profiles of the corresponding T oligos in solution, i.e., T oligos with fast blinking kinetics had low fluorogenic profiles in solution and vice versa (Figure 2).

[0235] We then analyzed the focus kinetics profiles of each of the eight foci for complementary and mismatched T oligos immobilized on a biotin-streptavidin-coated coverslip, allowing multiple blinking events to be captured from individual foci throughout the capture period (1000 frames, 20 ms / frame) without dissociating the T oligos from the coverslip surface. Foci were measured with blinking complementary T oligos with τ in the range of tens of milliseconds to seconds, compared to mismatched T oligos, whose on-times τ rarely exceeded 100 ms (Figure 12). The average number of blinking events during the total duration of exposure was also lower for matched T oligonucleotides (1.8) compared to mismatched T oligonucleotides (4.6). This shows two exemplary metrics that can be used to deconvolute the identity of T oligonucleotides.

[0236] Finally, a mixture of streptavidin-coated polystyrene beads conjugated with mismatched and complementary T oligos was immobilized in a single well on a BSA / biotin-coated glass and imaged in TIRF mode in the presence of 100 nM FRET oligo probe. For each measurement, the fluorescence from a single focus was recorded 1,000 times with 20 ms exposure. As seen previously with T oligos immobilized on glass coverslips, foci with multiple blinking events during capture were identified, representing immobilized T oligos on the bead surface. Foci with longer τ could be identified on beads coated with complementary T oligos, compared to the short τ detected on beads coated with mismatched T oligos (Figure 9).

[0237] conclusion Systematic fluorogenicity profiling of 90 FRET oligo / T oligo pairs in solution, generated by introducing one to six mismatches at random positions in a fully complementary T oligo, showed that the number of mismatches correlated with the fluorogenicity of the pair. That is, the more mismatches the T oligo contained, the less fluorogenic the FRET oligo was upon hybridization to the T oligo, presumably by destabilizing the bond between the FRET oligo and the T oligo. We also found that the position of the mismatch influenced the extent to which the fluorogenic profile of the FRET oligo was affected. Mismatches in the central part of the T oligo sequence appeared to have a greater impact on the fluorogenicity of the FRET oligo. Furthermore, adjacent mismatches had a stronger effect, presumably by creating a larger ridge and thus further destabilizing the hybridization pair.

[0238] We next assessed whether it was possible to establish a correlation between the fluorogenic profile and blinking rate of a FRET oligo / T oligo pair, such that the on / off rate of a given pair could be predicted simply by profiling its fluorescence in solution. We analyzed the blinking profiles generated when the FRET oligo was hybridized to an immobilized T oligo. We selected six different T oligos that resulted in unique fluorogenic profiles upon pairing with the FRET oligo. We observed that T oligos that generated low fluorogenic profiles in solution upon pairing with the FRET oligo showed high and fast blinking rates when immobilized on a surface, whereas highly fluorogenic pairs showed low blinking rates. This suggested an inverse correlation between the fluorogenic profile and the blinking kinetic profile. This was further confirmed by analyzing the focal kinetic profiles of the FRET oligo when added to a fully complementary T oligo or a mismatched T oligo, this time immobilized on beads to avoid potential nonspecific blinking. Two different types of foci were identified, long and short τ foci, corresponding to hybridization of FRET oligos to perfectly complementary and mismatched immobilized T oligos, respectively. These results successfully demonstrated that focal dynamics profiles can be established for different FRET oligo / T oligo pairs imaged simultaneously.

[0239] Example 3 Example 3 - Spectrally Independent Multiplexed Imaging (SIMI) of six cellular targets on fixed cells T oligos (1 to 6, see Table 3) were covalently linked to six primary antibodies that recognize six different epitopes within cells to form six primary antibody-T oligo conjugates. [Table 4]

[0240] Commercially available human cancer cells were fixed and permeabilized (see Materials and Methods). The fixed and permeabilized samples were then contacted with a primary antibody-T oligo conjugate. The cells were washed to remove unbound antibody-T oligo conjugate. Two FRET oligos (A and B, see Table 3), both conjugated to a Cy5 fluorophore at the 5' end and a BHQ-3 dark quencher at the 3' end, were then incubated with the samples.

[0241] Each of the two FRET oligos can reversibly hybridize to an antibody-T oligonucleotide conjugate in its set, i.e., T oligos a-b can hybridize to FRET oligo A in set 1, and T oligos c-f can hybridize to FRET oligo B in set 2.

[0242] The samples were illuminated at the excitation wavelength of Cy5. For each FRET oligo, the quencher BHQ-3 suppresses the fluorescence emission from the emitter fluorophore Cy5 when it is not hybridized to one of the respective T oligos. Upon hybridization, this quenching is released, allowing detection of fluorescence at the hybridization site.

[0243] A high-speed CMOS sensor captures images of the sample as sequential images over time in seconds to minutes (Figure 14A). The fluorescence of each of the focal points is monitored over time, and a kinetic fluorescence profile is generated. The fluorescence profile (or one or more metrics thereof) at a particular focal point is used to determine the fluorescence kinetic profile of that focal point. In this example, the focal point is 2-3 pixels in diameter. The identity of each pixel is assigned to one of the FRET oligo / T oligo pairs or to the background.

[0244] Because each different FRET oligonucleotide T oligonucleotide pair produces a fluorescence kinetic profile that is unique to that pair, the fluorescence kinetic profile at each pixel (an example trace is shown in FIG. 14C) can be assigned an identity (e.g., to the particular T oligonucleotide that produced it). In this way, an identity is assigned to a pixel based on the fluorescence kinetic profile of the sample at that pixel (e.g., defined in terms of its at least one metric).

[0245] A super-resolution image was reconstructed from the accumulated frames using standard localization algorithms such as those found in ThunderSTORM (Ovesny et al. Bioinformatics 2014), QC-STORM (Li et al, Opt. Express 2019), and Picasso (Schnitzbauer et al, Nature Protocols 2017) software (Figure 14A). In this example, demultiplexing was performed on the super-resolution image (Figure 14B). However, demultiplexing is possible with or without super-resolution reconstruction. Target assignment was performed using a standard cluster-based mathematical approach on spatiotemporal metrics derived from the blinking profile associated with each FRET oligo / T oligo. Figure 14D shows a scatter plot of two extracted features (e.g., blink duration and off time) that can be demultiplexed (Figure 14D).

[0246] In this example, only one type of fluorophore (Cy5) is used to label the FRET oligos, so the set of FRET oligo and antibody-T oligonucleotide conjugate pairs shown in Table 3 form one set of pairs (group 1) that have the same "color" (i.e., they are observed through a single channel). In other examples, if additional groups of FRET oligos emit different "colors," further sets (e.g., group 2, group 3, ...) may be added in which the pairs produce a similar or identical profile to that seen in group 1, e.g., by using Cy3 as the fluorophore to label the FRET oligos. This additional level of multiplexing is deconvoluted by capturing images using multiple different imaging channels.

[0247] Materials and Methods Oligo Two FRET oligos were used, 15 and 20 nucleotides in length, respectively, both conjugated to a Cy5 fluorophore at the 5' end and a BHQ-3 dark quencher at the 3' end. T oligos were generated by introducing mismatches at random positions into the otherwise fully complementary sequences using a Python script. The sequences of the T oligos are shown in Table 3. T oligos and FRET oligos were purchased from IDT, resuspended in 100 μM TE buffer (1 M Tris-HCl, pH 8.0, 0.5 M EDTA pH 8.0), and stored in aliquots at -20°C.

[0248] Primary antibody conjugation For each target, 15 μg of commercial primary antibodies against 1) α-tubulin, 2) calnexin, 3) golgin-97, 4) Ki-67, 5) myosin IIB, and 6) Tomm-20 were used for conjugation with different T oligos, supplemented with 800 pmol of oligo per conjugation. The antibody conjugates were then purified on a 50 kDa Amicon Ultra-0.5 column and then stored at 4°C.

[0249] immunolabeling Cells were seeded in 18-well glass-bottom microslides (Ibidi) and fixed with 4% PFA when approximately 80% confluent. Cells were incubated for 60 min at room temperature in blocking buffer (1x PBS, 1% BSA, 0.1% Triton-X, 0.2 mg / ml sheared salmon sperm DNA) and then incubated overnight at 4°C with T oligo-antibody mix, each T oligo-antibody conjugate diluted 1 / 100–1 / 200 in 1x PBS, 1% BSA, 0.1% Triton-X, 0.1 mg / ml sheared salmon sperm DNA, 150 mM NaCl, 5 mM EDTA, 0.05% dextran sulfate.

[0250] Imaging Immunolabeled cells were incubated with 5 nM of FRET oligos diluted in imaging buffer (1x PBS, 10 mM Tris-HCl pH 8, 500 mM NaCl, 1 mg / ml glucose oxidase, 5 mg / ml glucose, 0.04 mg / ml catalase, 1 mM Trolox). 10,000 images were captured with a Nikon 60x 1.49NA TIRF objective at 10 ms / frame, 16-bit, 2-CMS gain mode and filtered with a Cy5 filter set with a Prime BSI Express sCMOS camera. Final super-resolution images were reconstructed using QC-STORM or Picasso software. Spatiotemporal metrics derived from the blinking profiles associated with each FRET oligo / T oligo were then demultiplexed to obtain the identity of individual targets from each channel using standard cluster-based mathematical approaches.

Claims

1. 1. A method for multiplexed fluorescence microscopy, comprising: a. contacting a fixed sample with a set of binding agent-T oligonucleotide conjugates to allow the binding agent to bind any binding partner present in the sample, the set comprising multiple binding agents with different specificities, the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated; b. contacting the sample and any binding agent bound as a result of step a with a FRET oligonucleotide; c. irradiating the sample with a wavelength that causes excitation of the emitter molecule of the FRET oligonucleotide; d. observing a fluorescence kinetic profile of said sample over time in one or more pixels at the emission wavelength of said FRET oligonucleotide emitter molecule; Including, The method, wherein the FRET oligonucleotide can hybridize to multiple T oligonucleotides in the set to form multiple pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within the set.

2. (A) the method further comprises calculating a distinguishable metric from the fluorescence kinetic profile and, optionally, assigning an identity to one or more pixels in the observed sample based on the metric calculated from the fluorescence kinetic profile of the sample at that pixel; (B) the unbound binder-T oligonucleotide conjugate is removed after step a; (C) the sample is re-fixed after step a to further immobilize any bound binding agents; (D) a) each T oligonucleotide in the set is 8 to 35 nucleotide units in length; and / or b) the FRET oligonucleotide is (i) longer than the Förster radius of the FRET oligonucleotide emitter molecule, and / or (ii) at least 12 nucleotide units in length; (E) each T oligonucleotide is capable of hybridizing to said FRET oligonucleotide, and each T oligonucleotide has 0-40% base pair mismatch with said FRET oligonucleotide; and / or (F) the binding agent is a proteinaceous molecule, optionally an antibody or a fragment thereof; The method of claim 1. (A) at least two metrics are calculated from the fluorescence kinetic profile and used to assign an identity to one or more pixels in the observed sample; and / or (B) at least one metric calculated or derived from the fluorescence kinetic profile of each distinct FRET oligonucleotide and T oligonucleotide pair, (i) the average time between each fluorescence emission; (ii) the average duration of said fluorescent emission; (iii) the incidence of fluorescent emission; (iv) the dissociation constant (Koff) of the binding between the FRET oligonucleotide and the T oligonucleotide, as deduced from the fluorescence kinetic profile; and (v) the association constant (K on ) of the binding between the FRET oligonucleotide and the T oligonucleotide, as inferred from the fluorescence kinetic profile and the concentrations of the components; Selected from the group consisting of: The method of claim 2.

4. The method of any one of claims 1 to 3, wherein the fluorescence kinetic profile is observed using a high frame rate imaging device capable of detecting single molecule blinking events.

5. (i) step a comprises contacting the sample with a set of binder-T oligonucleotide conjugates, the set comprising a first set of binder-T oligonucleotide conjugates and one or more subsequent sets of binder-T oligonucleotide conjugates; (ii) step b comprises contacting the sample and any binding agent bound as a result of step a with a first FRET oligonucleotide and one or more subsequent FRET oligonucleotides; The first FRET oligonucleotide can hybridize to a plurality of T oligonucleotides in the first set to form a plurality of pairs, and each of the subsequent FRET oligonucleotides can hybridize to a plurality of T oligonucleotides in its corresponding set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is (i) unique to that pair within each set, and (ii) unique to that pair within the set population. The method according to any one of claims 1 to 4. (A)(i) the first FRET oligonucleotide and the subsequent FRET oligonucleotide comprise emitter molecules having overlapping emission spectra, and optionally the same emitter molecule; and / or (ii) step a comprises contacting the sample with a plurality of sets of binder-T oligonucleotide conjugates, each set comprising a first set of binder-T oligonucleotide conjugates and one or more subsequent sets of binder-T oligonucleotide conjugates, wherein the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific for that set, and the step of observing the fluorescence kinetic profile of the sample comprises observing the fluorescence kinetic profile of the sample in a channel for each set, and optionally wherein for each set of binder-T oligonucleotide conjugates, the corresponding first FRET oligonucleotide and the subsequent FRET oligonucleotide comprise the same emitter molecule; or (B)(i) step a comprises contacting the sample with a plurality of sets of binder-T oligonucleotide conjugates, wherein for each set of binder-T oligonucleotides, the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide can be detected in a channel specific for that set; or (ii) Step a comprises: (I) one or more sets of binder-T oligonucleotide conjugates, wherein the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific for that set; and (II) one or more sets of binder-T oligonucleotide conjugates, wherein the emission spectrum of the emitter molecule in the corresponding FRET oligonucleotide for each set of binder-T oligonucleotides can be detected in a channel specific to that set. contacting the the step of observing the fluorescence kinetic profile of the sample includes observing the fluorescence kinetic profile of the sample in a channel for each set or for each group of sets; Optionally, (i) step a comprises contacting the sample with a plurality of sets of binder-T oligonucleotide conjugates, wherein for each set of binder-T oligonucleotide conjugates, the corresponding FRET oligonucleotide comprises an emitter molecule specific for each set; or (ii) Step a comprises: (I) one or more sets of binder-T oligonucleotide conjugates, wherein for each set of binder-T oligonucleotide conjugates, the corresponding FRET oligonucleotide comprises an emitter molecule specific for each set; and (II) one or more sets of binder-T oligonucleotide conjugates, wherein for each set of binder-T oligonucleotide conjugates, the corresponding first FRET oligonucleotide and the subsequent FRET oligonucleotide comprise the same emitter molecule. contacting the The step of observing the fluorescence kinetic profile of the sample includes observing the fluorescence kinetic profile of the sample in a channel for each set or for each group of sets. The method according to claim 5.

7. The method of any one of claims 1 to 6, wherein the or each set of binder-T oligonucleotide conjugates comprises from 2 to 25 different binder-T oligonucleotide conjugates.

8. The method of any one of claims 5 to 7, wherein the or each set of binder-T oligonucleotide conjugates comprises from 2 to 25 sets of binder-T oligonucleotide conjugates.

9. At least one set of binder-T oligonucleotide conjugates comprises: a. comprising one or both of the T oligonucleotides of sequence SEQ ID NO: 1 and SEQ ID NO: 2, and the corresponding FRET oligonucleotide is a FRET oligonucleotide of sequence SEQ ID NO: 7; and / or b. At least one set of binder-T oligonucleotide conjugates comprises at least one of the T oligonucleotides of the sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the corresponding FRET oligonucleotide is a FRET oligonucleotide of the sequence SEQ ID NO:8; The method according to any one of claims 1 to 8.

10. 1. A kit for multiplexed fluorescence microscopy comprising: a. a set of binding agent-T oligonucleotide conjugates, the set comprising multiple binding agents with different specificities, the sequence of the T oligonucleotide being unique to the binding agent to which it is conjugated; b. FRET oligonucleotide; wherein the FRET oligonucleotide is capable of hybridizing to a plurality of T oligonucleotides in the set to form a plurality of pairs, and dissociation and reassociation between each different pair generates a fluorescence kinetic profile that is unique to that pair within the set.

11. (A) the kit further comprises one or more additional sets of binder-T oligonucleotide conjugates, and one or more additional FRET oligonucleotides, each of the additional FRET oligonucleotides capable of hybridizing to a plurality of T oligonucleotides in its corresponding set to form a plurality of pairs, and dissociation and reassociation between each different pair produces a fluorescence kinetic profile within each set that is unique to that pair; and / or (B) at least one set of binder-T oligonucleotide conjugates comprises: a. comprising one or both of the T oligonucleotides of sequence SEQ ID NO: 1 and SEQ ID NO: 2, and the corresponding FRET oligonucleotide is a FRET oligonucleotide of sequence SEQ ID NO: 7; and / or b. At least one set of binder-T oligonucleotide conjugates comprises at least one of the T oligonucleotides of the sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the corresponding FRET oligonucleotide is a FRET-oligonucleotide of the sequence SEQ ID NO:8; The kit of claim 10.

12. A set of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides for producing a kit for multiplexed fluorescence microscopy, comprising: a. a set of T oligonucleotides; b. FRET oligonucleotide; Including, The set of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides, wherein the FRET oligonucleotide can hybridize to multiple T oligonucleotides in the set to form multiple pairs, and dissociation and reassociation between each different FRET oligonucleotide and T oligonucleotide generates a fluorescence kinetic profile that is unique to that pair within the set.

13. The set of binder-T oligonucleotide conjugates comprises: a. comprising one or both of the T oligonucleotides of sequence SEQ ID NO: 1 and SEQ ID NO: 2, and the corresponding FRET oligonucleotide is a FRET oligonucleotide of sequence SEQ ID NO: 7; or b. The set of binder-T oligonucleotide conjugates comprises at least one of the T oligonucleotides of the sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the FRET oligonucleotide is a FRET oligonucleotide of the sequence SEQ ID NO:8; A set of binder-T oligonucleotide conjugates and corresponding FRET oligonucleotides for preparing a kit for multiplexed fluorescence microscopy according to claim 12.

14. 13. A method for preparing the kit of claim 11, comprising providing a set of T oligonucleotides, and a set of binding agents and corresponding FRET oligonucleotides, and conjugating the set of T oligonucleotides of claim 12 with the set of binding agents to form a set of binding agent-T oligonucleotide conjugates, wherein the set of binding agent-T oligonucleotide conjugates comprises a plurality of binding agents with different specificities, and the sequence of the T oligonucleotide within the set is unique to the binding agent to which it is conjugated.

15. A method for designing a set of T oligonucleotides and FRET oligonucleotides for use in the method of any one of claims 1 to 9, comprising the steps of: a. selecting a FRET oligonucleotide sequence at least 12 nucleotides in length; b. Obtaining one or more sequences at least 8 nucleotides in length that are complementary to the FRET oligonucleotide sequence; c. generating a plurality of potential T oligonucleotide sequences, each of which differs by at least one nucleotide from the sequence complementary to the FRET oligonucleotide; d. selecting at least two T oligonucleotide sequences based on the ability of the at least two T oligonucleotide sequences to generate fluorescence kinetic profiles that differ from one another; The method comprising:

Images